CN1182868C - 有效地作为无毒口服佐剂的突变肠毒素 - Google Patents
有效地作为无毒口服佐剂的突变肠毒素 Download PDFInfo
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- CN1182868C CN1182868C CNB951958380A CN95195838A CN1182868C CN 1182868 C CN1182868 C CN 1182868C CN B951958380 A CNB951958380 A CN B951958380A CN 95195838 A CN95195838 A CN 95195838A CN 1182868 C CN1182868 C CN 1182868C
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Abstract
在此提供用于大肠杆菌热不稳定肠毒素的一种新突变形式的方法和组合物,这种肠毒素已丧失毒性但仍保留其免疫学活性。这种肠毒素与不相关抗原联合使用,当部分作为口服疫苗制剂施用时,获得对所述抗原增强的免疫应答。
Description
在本说明书中所述的研究由美国海军部分资助,授与号N00014-83-K-0192。政府对本发明享有一定权利。
1.发明领域
本发明涉及大肠杆菌热不稳定肠毒素(LT)的一种遗传学上独特突变型,及其作为一种口服佐剂用于诱导粘膜和血清抗体。特别地,该突变LT被一个氨基酸取代而修饰,使其失去固有的毒性,但保留了该分子完整的佐剂特点。
2.发明背景
微生物病原体可以通过几种机制中的一种感染宿主。它们可以通过外伤引起的外皮破裂进入,可以被载体传导而导入或与粘膜表面相互作用。大多数人病原体通过最后一种机制,即与粘膜表面相互作用后始发疾病。通过这种机制作用的细菌和病毒病原体首先与粘膜表面接触,它们可以在那儿附着,然后定居或被特化的表皮中遮盖派伊尔结及其它淋巴滤泡的吸收细胞(M细胞)摄取〔Bockman及Cooper,1973,Am.J.Anat.136:455-477;Owen et al.,1986,J.Infect.Dis.153:1108-1118〕。进入淋巴组织的生物体可在淋巴滤泡里被容易地杀死,因此作为提供给滤泡中免疫细胞的抗原(如
霍乱弧菌)激起可能的保护免疫应答。或者经过局部防御机制仍能存活的病原生物体可能从滤泡传播并继而引起局部的或系统疾病(即免疫受损害宿主中的
沙门菌属细菌,脊髓灰质炎病毒,轮状病毒)。
针对感染生物体的特异毒力决定簇的分泌IgA(sIgA)抗体在总体粘膜免疫方面起重要作用〔Cebra et al.,1986,In:Vaceines 86,Brown et al.,(ed.)Cold Spring Harbor Laboratorg,New York.p.p129-133〕,在许多情况下,通过刺激产生针对感染生物体的相应毒力决定簇的粘膜sIgA水平,来预防粘膜表面的初始感染是可能的,分泌IgA可以通过封闭附着和/或定居,中和表面作用毒素或防止宿主细胞的侵袭来防止病原体与粘膜表面的初始相互作用。虽然已经做了广泛的研究来确定,细胞介导的免疫及血清抗体在抵抗感染因子的保护作用方面的作用,对sIgA的调节,诱导和分泌却知之甚少。非肠道施用灭活的完整细胞和完整病毒的制剂对引发保护性血清IgG和抵抗在发病机理中具有明显血清期的生物体(即
伤寒杆菌,B肝炎)的延迟型超敏反应是有效的。然而,非肠道疫苗对引发粘膜sIgA反应无效,并对与粘膜表面相互作用但不侵入的细菌(如霍乱弧菌)无效。但最近有这样的证据,即非肠道施用的疫苗可能至少对一种病毒有效,它是首先与粘膜表面相互作用的轮状病毒〔Conner et al.,1993,J.Virol.67:6633-6641〕。推测保护作用来自抗原特异性IgG渗出到粘膜表面来中和病毒。因此,同时刺激血清和粘膜抗体的机制对有效疫苗是重要的。
如果抗原提呈到优先起始IgA B-细胞形成的派伊尔结中的T、B淋巴细胞和辅助细胞,则口服免疫对诱导特异sIgA反应有效。派伊尔结含有介导B-细胞直接从IgM细胞同型转换到IgA B-细胞的辅助T(TH)-细胞。该结还含有启动终未B-细胞分化的T-细胞。已接触抗原的B-细胞然后迁移到肠系膜的淋巴结并进行分化,进行胸导管,然后进入总循环,并播种到机体所有的分泌组织,包括肠固有层和呼吸道。然后由成熟的浆细胞产生IgA,IgA与结合在膜上的分泌成分复合,转运到能与侵入病原体相互作用的粘膜表面上〔Strober andJacobs,1985,In:宿主防御机制的进展,第4卷,粘膜免疫Gallin andFauci(ed.),Raven Press,New York.p.p.1-30;Tomasi and Plaut,1985,In:宿主防御机制的进展,第4卷,粘膜免疫Gallin and Fauci(ed.),Raven Press,New York p.p.31-61〕。这种共同的粘膜免疫系统的存在部分解释了对抵御通过首先与粘膜表面相互作用而始发感染的病原生物体起保护作用的活口服疫苗和口服免疫的可能性。
已经开发许多用于口服免疫的方法,包括利用细菌的减毒突变体(即沙门菌属细菌)作为异种抗原的载体(Cārdenas and Clements,1992,Clin.Microbiol.Rev.5:328-342;Clements et al.,1992,In:重组DNA疫苗:合理性及策略Isaacson(ed.),Marcel Decker,New York p.p.293-321;Clements and Cardenas,1990,Res.Microbiol.141:981-991;Clements and El-Morshidy,1984,Infect.Immun.46:564-569〕。将抗原胶囊化到由聚DL-丙交酯-乙交酯(PGL),蛋白样聚合物-类蛋白组成的微球中〔Sanitago et al.,1993,Pharma-Ceutical Research 10:1243-1247〕,明胶胶囊,不同的脂质体制剂〔Alving et al.,1986,Vaccine 4:166-172;Garcon and Six,1993,J.Immunol.146:3697-3702;Gould-Fogerite and Mannino,1993,In:脂质体技术第二版Vol.III,Gregoriadis(ed.)〕,吸附到毫微颗粒上,使用亲脂免疫刺激复合物(ISCOMS)〔Mowat and Donachie,1991,Immunology Today 12:383-385〕,以及加入具有已知佐剂性质的细菌产物〔Clements et al.,1988,Vaccine 6:269-277;Elson,1989,Immunology Today 146:29-33;Lycke and Holmgren,1986,Immunology 59:301-308;Lycke et al.,1992,Eur.J.Immunol.22:2277-2281〕。两种具有最大可能作为口服佐剂的细菌产物是由各株
霍乱弧菌产生的霍乱毒素(CT),和由
大肠杆菌的一些肠毒性株产生的热不稳定肠毒素(LT)。虽然LT和CT具有许多相同的特点,但它们虽然是不同的分子,具有使它们独一无二的生化和免疫学差异。
霍乱的大量腹泻是一种强效力外肠毒素的结果,它引起腺苷酸环化酶的激活,接着使细胞内环3′-,5′-腺苷单磷酸酯(cAMP)水平升高。霍乱肠毒素(CT)是一个84,000道尔顿,由两个主要的,非共价结合,免疫学上不同的区域或结构域(“霍乱毒素-A”和“霍乱毒素-B”)组成的多聚蛋白质〔Finkelstein and Lospalluto,1969,J.Exp.Med.130:185-202〕。其中该56,000道尔顿区域或霍乱肠菌素负责毒素与宿主细胞膜受体GM1(半乳糖基-N-乙酰氨基半乳糖基saminyl-(sialyl)-半乳糖基-葡萄糖基神经酰胺)的结合,该受体基本上在所有真核细胞的表面被发现。霍乱肠菌素由五个非共价结合的亚基组成,而A区域(27,000道尔顿)负责毒素多种生物学作用。
CT的两个亚基与该分子免疫学性质之间的关系曾经是大量辨论的原因。一方面,当CT口服施用时,它是一种能同时激发血清和粘膜抗毒素抗体反应形成的很好的免疫原。此发现并不新颖,因为已知霍乱病人在从临床霍乱渐愈期间抗毒素抗体的滴度逐渐升高〔Finkelstein,1975.Curr.Top.Microbid Immunol.69:637-196〕。研究该反应本质的人们的一个重要发现是现实的CT不像其它大多数蛋白抗原,不诱导对自身的口服耐受性〔Elson and Ealding,1984,J.Immunol.133:2892-2897;Elson and Ealding,1984,J.Immunol.132:2736-2741〕。当只喂给小鼠B-亚基时,也发现这是真的,通过在孟加拉国进行的霍乱疫苗现场试验证实此观察,其中B-亚基与杀死的完整细胞联合使用的口服免疫引起粘膜和系统的抗毒素抗体反应〔Svennerholm et al.,1984,J.Infect.Dis.149:884-893〕。
CT除了是一种有效的口服免疫原,还具有一些其它已报道的免疫学性质。如上面指出的,Elson和Ealding〔Elson and Ealding,1984,J.Immunol.133:2892-2897〕观察到口服CT不诱导对自身的耐受。而且同时口服CT和一种可溶蛋白抗原钥孔形血蓝素(KLH)导致抗CT和KLH分泌IgA反应的形成,并废除对KLH口服耐受的诱导。这些发现继而被Lycke和Holmgren〔Lycke and Holmgren,1986,Immunology59:301-308〕证实和扩展。当人们试图确定CT的A和B亚基对该分子佐剂性质的作用时发生混乱。下列由Elson概括的观察〔Elson,1989,Immunology Today 146:29-33〕是那些混乱的基础:
·CT不诱导对自身的口服耐受〔Elson and Ealding,1984,J.Immunol.133:2892-2897〕
·CT-B不诱导对自身的口服耐受〔Elson and Ealding,1984,J.Immunol.133:2892-2897〕
·CT可防止对其它抗原的耐受的诱导,当CT被同时施用并用作那些抗原的一种佐剂时〔Elson and Ealding,1984,J.Immunol.133:2892-2897;Lycke and Holmgren,1986,Immunology 59:301-308〕。
·CT可作为CT-B的佐剂〔Elson and Ealding,1984,J.Immunol.133:2892-2897〕
·热凝集CT几乎没有毒性,但是一种有效的口服免疫原〔Pierce etal.,1983,Infect.Immun.40:1112-1118〕。
·CT-B可以在传统的半抗原-载体结构中作为一种免疫原“载体”〔Cebra et al.,1986,In:Vaccines 86,Brown et al.,(ed.),ColdSpring Harbor Laboratory,New York.p.p.129-133;McKenzieand Halsey,1984,J.Immunol,133:1818-1824〕。
一些研究人员已经从这些发现断定B-亚基一定具有一些固有的佐剂活性。Cebra等〔Cebra et al.,1986,In:Vaccines 86,Brown etal.,(ed.),Cold Spring Harbor Laboratory,New York.p.p.129-133〕,Lycke和Holmgren〔Lycke and Holmgren,1986,Immunology59:301-308〕和Liang等〔Liang et al.,1988,J.Immunol.141:1495-1501〕会辩论反对该结论。Cebra等〔Cebra et al.,1986,In:Vaceines 86,Brown et al.,(ed.)Cold Spring Harbor Laboratorg,New York.p.p129-133〕证明纯化的CT-B当十二指肠内给药时对提高派伊尔结中特异抗霍乱毒素B-细胞的频率有效,但与CT相反,不产生大量的提呈IgA的B-细胞。Lycke和Holmgren〔Lycke andHolmgren,1986,Immunology 59:301-308〕通过测量口服免疫小鼠固有层中分泌免疫球蛋白的细胞,比较了CT和CT-B增强肠粘膜对KLH免疫反应的能力。在他们的系统中,未发现对被试任何剂量的B-亚基反应的产生抗-KLH细胞的增加。最终,Liang等〔Liang et al.,1988,J.Immunol.141:1495-1501〕发现当CT-B连同灭活的Sendai病毒口服给药时没有佐剂作用。
观察到分离B-亚基佐剂活性的地方,典型地具有两个原因之一。首先,传统制备B-亚基的方法是在变性剂(即盐酸胍或蚁酸)存在下通过凝胶过滤,将全毒素进行层析分离。然后收集分离的亚基,除去变性剂。用此技术制备的B-亚基一定受到微量A-亚基的污染,以致于复性时重建了少量全毒素。第二个原因与免疫载体的定义有关。与其它许多可溶性蛋白一样,B-亚基可以作为一种免疫载体向免疫系统提呈抗原。假如那些抗原足够小以致不具有好的致免疫性,它们可以传统的半抗原-载体构型制成致免疫的。同样,有与B-亚基相关的“假设”免疫增强,特别对于口服施用,因为B-亚基与上皮细胞表面结合,并可能固定于一种附着抗原被肠相关淋巴组织加工。然而,对这种抗原稳定化机制的任何可能的优点,都可以被跨越非免疫相关组织的抗原的分布所抵消,即小肠上皮细胞的表面。在粘膜反应情况下,免疫学相关部位是派伊尔结,特别对依赖于抗原特异性T细胞的B细胞激活〔Stroberand Jacobs,1985,In:宿主防御机制的进展,第4卷,粘膜免疫Gallinand Fauci(ed.),Raven Press,New York.p.p.1-30;Tomasi andPlaut,1985,In:宿主防御机制的进展,第4卷,粘膜免疫Gallin andFauci(ed.),Raven Press,New York.p.p.31-61;Brandtzaeg,1989,Curr.Top.Microbio.Immunol.146:13-25〕。因而,该条件胜任在派伊尔结中发生的从IgM细胞到IgA B-细胞的同型转变。位于上皮细胞表面的抗原可能有助于诱导B细胞增殖的抗原,因为亚类阳性绒毛上皮细胞可能在分泌部位作为抗原提呈细胞用于T细胞激活,因此增加细胞因子产生,终末B细胞分化,增加分泌成分的表达,并增加IgA特异性抗原的外部转运〔Tomasi,T.B.,and A.G.Plaut.1985,In:宿主防御机制的进展,第4卷,粘膜免疫Gallin and Fauci(ed.),Raven Press,New York.p.p.31-61〕这些事件的关系尚未明确地解释为B-亚基作为其它抗原的载体,术语:“佐剂”的使用似乎对这种作用不合适。
已经清楚分子的佐剂性质属于全毒素,其中B-亚基是受体识别所需要的,并利于A-亚基渗透进入细胞。A-亚基也是佐剂活性所必需的,推测其具有ADP-核糖基化的活性,并能增加细胞内cAMP水平(见下)。B-亚基单独可以作为其它抗原的载体,因为当它们与那些抗原结合时,能够被固定化以由肠相关淋巴组织加工。
虽然LT和CT具有许多共同的特点,它们明显地是不同的分子,具有使它们独一无二的生化和免疫学差异,包括在核苷酸和氨基酸序列同源性20%的差异〔Dallus and Falkow,1980,Nature,288:499-501〕。两种毒素具有相同的亚基数目和排列,相同的生物学作用机制,并在许多体外试验中具有相同的比活〔Clements and Finkelstein,1979,Infect.Immun.24:760-769;Clements et al.,1980,Infect.Immun.24:91-97〕。
然而,这些分子之间存在不仅影响其外毒素性质还影响其作为佐剂能力的显著差异。不同于霍乱弧菌产生的CT,LT存在于相关细胞并只在细胞裂解过程中从大肠杆菌中释放〔Clements and Finkelstein,1979,Infect.Immun.24:760-769〕。CT一旦合成后就从弧菌分泌出来,并能被容易地从培养物上清中鉴定和纯化。所以,与CT相比,LT当刚从细胞分离时并非有完整的生物活性。与细菌毒素的A-B模型相一致,LT需要蛋白酶水解和二硫化物还原成为完全活性的。没有蛋白酶水解处理,酶活性A1部分不能与A2部分分离,也不能到达小肠上皮细胞基底层(basolateral)表面的靶底物(腺苷酸环化酶)。这对CT也是一样的,但该毒素在纯化过程中,暴露于培养物上清液中的蛋白酶完成该蛋白水解。因为LT不是完全生物活性的,在纯化过程中用体外生物学试验如Y-1肾上腺细胞试验或渗透因子试验难以鉴定。
分离材料激活中的差异导致LT和CT在生物系统中反应阈值的差异。例如,CT剂量为5-10μg诱导小鼠肠中可察觉的网流体分泌。LT在大于100μg水平诱导小鼠肠中可察觉的网流体分泌。在兔结扎的回肠袢中,差异是引人注目的和清晰的。而且,在灵长目动物,LT在被试的高达1mg的任何剂量均显示不能诱导流体分泌。这是报道的CT诱导人阳性流体运动所用量的200倍。当LT暴露于具有胰蛋白酶样特性的蛋白水解酶时,该分子在任何生物测试系统均与CT难区分。这被Clements和Finkelstein清楚地证明〔Clements and Finkelstein,1979,Infect.Immun.24:760-769〕。
除了上面报道的差异外,LT对含有糖类的基质具有不寻常的亲合性。特别是分子量为90,000的LT,从葡聚糖柱(葡萄糖)洗脱下的表观分子量为45,000,从琼脂糖柱(半乳糖)洗脱下的表观分子量为0。这说明它与含半乳糖的基质结合,并且可以通过使用半乳糖以纯的形式从那些基质洗脱下来。LT不仅与用于纯化的柱上的琼脂糖结合,而且更重要地与其它含半乳糖的生物分子结合,包括糖蛋白和脂多糖。LT的凝集素样结合性质导致LT比CT在哺乳动物细胞上有更广泛的受体分布,CT只与GM1结合。这可能部分说明这两种分子诱导不同辅助T淋巴细胞反应能力上差异的原因〔McGhee et al.,1994,Mucosal ImmunologyUpdate,Spring 1994,Raven Press,New York,p21〕。
McGhee等报道的这些研究中〔McGhee et al.,1994,MucosalImmunology Update,Spring 1994,Raven Press,New York,p21〕,显示用疫苗对小鼠的口服免疫,如将CT作为粘膜佐剂的破伤风类毒素(TT),通过产生IL-4和IL-5而非IL-2和INF-gamma的TH细胞证明,在派伊尔结和脾脏中选择性地诱导TH2型细胞〔对细胞因子网更全面的综述见Arai et al.,1990,Ann.Rev.Biochem.59:783-836〕。
重要地,当CT被用作粘膜佐剂时,除IgA应答外它还增强抗原 特异性IgE应答。由于预料到诱导速发型超敏反应这种IgE应答的增强严重地危及CT作为粘膜佐剂的安全性。与此相反,当LT用作口服粘膜佐剂时,同时诱导TH1和TH2细胞,而且主要是抗原特异性IgA应答,没有IgE应答。
这两种分子还具有许多免疫学差异,如通过免疫扩散研究〔Clements and Finkelstein,1978,Infect.Immun.21:1036-1039;Clements and Finkelstein,1978,Infect.Immun.22:709-713〕,体外中和研究和在接受B-亚基完整细胞霍乱疫苗的志愿者中对LT相关的大肠杆菌腹泻的部分预防〔Clements et al.,1980,J.Infect.Dis 158:372-377〕所证明。
总之,这些发现表明尽管LT和CT表面上相似,它们是独一无二的 分子,并且LT是一种实用的口服佐剂而CT不是。
对LT佐剂性质的证明产生于本发明的一位发明者研究LT对口服抗原耐受形成的影响。尚不清楚如果给出两分子间观察到的差异,是否LT也影响口服耐受的诱导,或者LT起已证明的CT所起的佐剂作用。因此,本发明者检测了一些参数,包括LT对OVA口服耐受的作用,在观察的反应中LT两亚基的作用,改变时间和施用途径的情况下输送LT影响,先暴露于OVA对LT影响OVA耐受的能力的影响,LT作为佐剂与两种不相关抗原的使用,及免疫途径对抗OVA反应的影响。从这些研究得到的结果总结如下〔Clements et al.,1988,Vaccine 6:269-277;Clements et al.,1988,Abstract No.B91,88th Ann.Mcet.Am.Soc.Microbiol〕:
1.同时施用LT和OVA表明可防止诱导OVA耐受,并对OVA及PBS致敏的动物,分别增强血清抗-OVA IgG反应30-90倍。该作用被确定为毒素具酶活的A-亚基的作用,因为B-亚基单独不能影响耐受性诱导。
2.先用OVA致敏再喂以LT的动物比那些初始免疫中同时施用LT和OVA的动物,形成明显较低的血清IgG和粘膜的IgA抗-OVA反应,表明先暴露于抗原降低LT影响耐受的作用及其作为佐剂的能力。一旦耐受性建立,LT不能废除之。还发现对CT也是真的,当动物先用OVA免疫再口服卵清蛋白加CT,而且提供了一些对有益观察的洞悉,即当使用这些佐剂时不增加对非靶向摄入抗原的抗体反应。
3.第一次暴露于抗原时,只接受一次LT的动物,其血清IgG和粘膜IgA应答与经三次OVA/LT致敏的反应相同,表明应答的许诺发生在早期并在第一次暴露于抗原时,还证明对应答主要是针对血清IgG还是粘膜IgA,可以通过是否施用胃肠外加强剂量来控制。
4.如果动物没有预先用两种抗原中任何一种免疫,同时施用LT和两种可溶性蛋白抗原导致抗两种抗原的血清和粘膜抗体的形成。这是一项重要的发现,因为LT作为佐剂的一种可能的应用是形成对复合抗原如杀死的细菌或病毒的粘膜抗体的形成,在这里对多种抗原反应的能力是重要的。
Tamura等〔Tamura et al.,U.S.Patent No.5,182,109〕的研究证明鼻内施用LT和/或CT增加对共同施用的抗原的抗体滴度。然而,在Tamura等中没有教导口服给药时,这些毒素可诱导保护性免疫应答。
显然,LT具有明显的免疫调节能力,不论作为预防特异抗原耐受的诱导的一种手段,还是作为佐剂用于口服抗原,而且引发产生抗施用抗原的血清IgG和粘膜IgA。这提出了针对各种病原体,包括口服施用死的或减毒剂或特异剂的相关致病力决定簇的有效免疫方案的可能性。然而,该“毒素”当被如肠蛋白水解酶进行蛋白水解作用裂解时,或口服施用足够高浓度时能刺激网腔(net lamemal)分泌反应的事实会妨碍其能力的调查或阻止其在合适条件下的应用。如果LT在不减少其佐剂性质下可以“去毒”,这个问题可以解决。这令人们意识到这可能是怎样实现,有必要进一步分析LT和CT的作用机制以及这些分子结构上和功能上的关系。如前面指出的,LT和CT以具有A和B成分的多亚基毒素合成。毒素与宿主细胞膜受体初始相互作用后,B区域利于A-亚基渗透穿过细胞膜。巯基还原时,A成分分裂成两个较小的多肽链。其中A1片段催化上皮细胞基质层表面腺苷酸环化酶复合物中兴奋性GTP结合蛋白(Gs)的ADP-核糖基化,而且这导致细胞内cAMP水平升高。此cAMP增加引起水和电解质分泌到小肠中,它通过与两种cAMP-敏感的离子转运机制相互作用,包括1)跨越绒毛状上皮细胞纹绿的NaCl共转运,和2)通过滤泡细胞分泌致电Na+依赖的Cl-〔Field,1980,In:Secretory diarrhea,Field et al.(ed.),Waverly Press,Baltimore p.21-30〕。A亚基还是与这些毒素的免疫增强相关的主要部分。该亚基然后成为可能的操作靶子以分开分子的毒性和免疫学功能。Lycke等〔Lycke et al.,1992,Eur.J.Immunol.22:2277-2281〕最近的报道搞清楚影响该毒素ADP-核糖基化酶活性的变化和改变增加细胞内cAMP水平的能力的变化也阻止该分子的佐剂作用。因此必须开发另一种去毒方法。
3.发明概述
本发明基于惊奇的发现,即失去毒性作用和缺乏ADP-核糖基转移酶活性的LT突变形式仍保留其作为免疫佐剂的活性。LT的突变形式通过单个氨基酸取代Arg192-Gly192而与野生型不同,它使一个胰蛋白酶敏感位点不敏感。蛋白水解酶位点的丧失防止A亚基的蛋白水解加工成其毒性形式。天然LT当第一次从细菌分离时没有毒性,但当暴露于蛋白酶如哺乳动物小肠中发现的,则具有潜力为完全有毒的。LT的突变形式不再具有由于蛋白水解激活而变为有毒的潜力。该突变LT(此后称mLT)保有增强针对与LT或mLT不相关抗原的动物免疫反应(如IgG,IgA)的能力且无毒副作用。实验证据显示mLT具有为口服抗原作佐剂的用途;这种施用导致对与mLT一起施用的抗原的血清IgG和/或粘膜IgA的产生。本发明提供一种在宿主中诱导对任何口服抗原的血清和/或粘膜免疫反应的方法,它包括向宿主施用有效量的mLT并口服有效量的该抗原。优选地该抗原和mLT优选在开始时同时施用。
本方法和组合物提供一种改善的口服免疫方式,用于产生抗病原微生物的血清和粘膜抗体。针对渗透或侵入并穿过粘膜表面的病原微生物的IgA抗体反应的产生能够指向那个表面,而能够产生显著的血清抗体反应以防止病原微生物的感染,其中,该病原微生物正是血清抗体所保护的。本发明对任何特异抗原是有用的,在那里特异的中和抗体反应将有益于消除与该抗原相关的生理或疾病状态。
本发明还提供一种组合物,可用作抗表达霍乱样肠毒素的肠毒素细菌生物体的一种疫苗成分及其使用方法。
本发明还提供在这些方法中有用的一种组合物。该组合物包含有效量的mLT与有效量的抗原。
4.附图简述
通过参考下列对本发明的详细描述,本发明特殊实施方案的实施例以及附图,会更全面地了解本发明,其中:
图1.质粒pBD94的图谱,它在lac启动子的控制下编码A和B亚基。质粒pBD95在A亚基的第192个氨基酸残基处有一个碱基替换,编码甘氨酸而非精氨酸,它保留了阅读框但消除了蛋白酶水解位点。图中显示对应于胰蛋白酶敏感区的氨基酸序列和Arg192-Gly192氨基酸替换位点。
图2.图解证明随CT量的增加ADP-ribosylagmatine水平的剂量依赖性增加。
图3.小鼠喂以125μg的天然LT而非125μg的mLT后的流体积蓄。肠-屠体比定义为肠重量除以剩余的屠体重量。
图4.mLT作为免疫佐剂的能力。图4A,mLT诱导对应于OVA的血清IgG的能力。图4B,mLT诱导对应于OVA的粘膜sIgA的能力。
图5.mLT保留了防止对LT口服耐受诱导的能力的实验证明。图5A,mLT诱导对应于LT的血清IgG的能力。图5B,mLT诱导对应于LT的粘膜sIgA的能力。
5.发明详述
本发明包含用于促进抗原的粘膜和血清抗体产生的组合物及其应用方法,这些抗原同遗传学改造的细菌毒素同时口服施用。改造的毒素是大肠杆菌热不稳定肠毒素(LT)的一种形式,它是通过基因工程丧失其胰蛋白酶敏感部位使分子无毒,而且出人意料地保留其作为免疫佐剂的能力。突变的LT在此称做“mLT”。本发明基于一个出人意料和令人吃惊的结果的发现,即mLT与LT一样是一种有效的免疫佐剂。mLT不再有ADP-核糖基化的酶活性,因为A亚基不能被蛋白水解加工。与Lycke及其同事发表的研究不同,他们弄清影响LT的ADP-核糖基化活性的改变也阻止该分子的免疫佐剂作用〔Lycke et al.,1992,Eur.J.Immunol.22:2277-2281〕,目前描述的mLT虽然如实施例中所证明的不具有ADP-核糖基化活性,但保留了免疫佐剂活性。
这里描述的大肠杆菌热不稳定肠毒素的新突变形式表现为佐剂,并激发产生被递送的抗原的血清IgG和粘膜sIgA。这项令人惊奇的发现的用途在于佐剂有效量的mLT可用于抗各种病原体的有效免疫方案,包括口服有效量的mLT佐剂与杀死的或减毒的病原体或特异病原体的相关毒力决定簇,不必担心口服CT或LT所带来的真正或潜在的毒副作用。
本发明废弃了现有技术,因为本发明可以用于各种免疫学应用,在此CT、LT或它们的亚基可能已被使用,但现在使用的mLT没有真正的或潜在的与其使用相关的副作用,如腹泻。与LT相比,虽然当首次从细菌分离时LT无毒,但当暴露于如在哺乳动物肠中发现的蛋白酶时有可能是完全有毒的,mLT不可能由于蛋白酶水解激活变为有毒。
本发明的另一个实施方案是作为抗表达霍乱样毒素的肠毒生物体的疫苗的一种成分。本发明者已指出mLT在给药时(见下)不受口服诱导的免疫耐受,因此mLT能起作用而且很有希望作为直接抗肠毒生物体的疫苗的一种成分。现有技术提供抗表达霍乱样毒素生物体的疫苗,包含杀死的完整细胞和毒素的B亚基。通过在疫苗中用mLT替换B亚基,该疫苗在两个不同方面得以改进。第一,具有A和B亚基的mLT将不仅诱导对B亚基的免疫反应,而且还诱导对A亚基的免疫反应。这为有效中和提供了更多的表位。第二,mLT固有的佐剂活性会增强抗疫苗中杀死的完整细胞成分的免疫应答。
而且,其它研究者〔Hase et al.,1994,Infect.Immun.62:2051-3057〕已指出A亚基通过改变ADP-核糖基化酶活性的活性部位以修饰使其不再有毒(与作为本发明主题的蛋白水解部位相反),便能够诱导针对野生型A亚基的免疫应答。然而,现在这样改造的A亚基缺乏免疫佐剂活性,所以不如mLT更有希望成为疫苗成分。
再者,因为抗mLT的抗体与LT和CT交叉反应,mLT可用于直接抗多种类型表达霍乱样毒素的肠毒细菌生物体,如埃希氏杆菌属和弧菌属细菌的疫苗。
5.1 mLT的生产
野生型LT毒素被发现于能产生此毒素的产肠毒素的大肠杆菌株的天然存在质粒编码。本发明者先前已从叫做H 10407的大肠杆菌人分离物中克隆了LT基因。该亚克隆由插入pBR322质粒PstI位点的H 10407肠毒素质粒中的5.2kb DNA片段组成〔Clements et al.,1983,Infect.Immun.40:653〕。此叫做pDF82的重组质粒已被广泛鉴定并在天然LT启动子的控制下表达LT。此过程的下一步是将LT基因置于强启动子的控制之下,此处的pUC 18质粒的lac启动子。通过分别分离LT-A和LT-B的基因并将它们重组在载体质粒的一个盒中完成。这是一个重要步骤,因为它允许合理数量的LT的纯化并衍生为随后分析的突变体。该叫做pBD94的质粒图解见图1。
CT和LT都用连接A1和A2片段的胰蛋白酶敏感肽键合成。此肽键必须是缺口的,以使该分子“有毒”。对白喉毒素,原型A-B毒素和各种其它细菌毒素也一样。不论通过细菌蛋白酶或肠腔中的蛋白酶,如果A1-A2键没有去掉,则A1片段不能达到其小肠上皮细胞基质层表面的靶上。与CT相比,LT当刚从细胞分离时不具完全的生物学活性。LT还需要蛋白水解成为完全活性的,而且蛋白水解激活不发生于细菌内部。所以,改变分子的毒性并不影响ADP-核糖基化酶活性的一种方法可能是通过基因操作,去掉连接A亚基的A1和A2成分的胰蛋白酶敏感氨基酸。如果该分子不能被蛋白水解切割,它就无毒。本领域的技术人员会预言该分子可能保持其ADP-核糖基化酶活性并最终保持其佐剂功能。
图1表示分开A1和A2片段的二硫包裹区域的序列。该区域中有一个精氨酸残基,它被认为是激活该分子毒性所必需的切割位点。该区域通过定位致突变被改变,以使分子对蛋白水解消化不敏感,最终无毒。
定位致突变通过将单链DNA与合成的寡核苷酸杂交完成的,该寡核苷酸除接近中央的失配区域外与单链模板互补。就是这个区域含有想要的一个或多个核苷酸改变。与单链靶DNA杂交后,该寡核苷酸用DNA聚合酶延伸以产生一个双链结构。缺口然后用DNA连接酶封口,针双链体结构转化到大肠杆菌宿主中。由于DNA的半保留复制模式,使用此方法的突变体理论产率是50%。实际上,产率更低。然而有些方法能改进产率并用于选择寡核苷酸定向突变体。采用的系统利用第二突变寡核苷酸,在两次突变策略中产生改变的限制性部位。
下一步是将Arg替换成另一氨基酸(即,GGA=甘氨酸替换AGA=精氨酸),虽然消除了蛋白水解位点但保留了阅读框。然后通过琼脂糖亲合层析,从已经测序证明的一个突变体(pBD95)纯化mLT。其它纯化方法对本领域的技术人员是显而易见的。此叫做LT(R192G)的突变LT通过SDS-聚丙烯酰胺凝胶电泳检测对胰蛋白酶敏感键的修饰。样品暴露或不暴露于胰蛋白酶进行检测并与天然(未修饰的)LT相比较。当与胰蛋白酶温育时mLT不裂解为A1和A2,因而表明去掉了对蛋白酶的敏感性。
5.2 mLT和非相关抗原的给药形式
根据本发明,mLT能与任何生物学上相关抗原和/或疫苗联合用药,以获得对所说抗原和/或疫苗增强的免疫反应。在优选实施方案中,mLT和抗原以含有有效量的mLT和有效量的抗原的药物组合物被同时施用。给药形式为口服。mLT和抗原的相对量会依照被采用抗原的本性和被免疫动物的种类而变化。在一个实施方案中,先施用mLT和抗原,再用相关抗原加强免疫。在另一个实施方案中不施用加强免疫。加强免疫的时间选择依赖于抗原和被处理的种类。通过常规实验,容易确定剂量范围以及对被施用种类和抗原的加强免疫时间选择的变更。加强免疫可只有抗原或与mLT合用。加强免疫的给药形式可口服、鼻腔或胃肠外,但如果在加强免疫中用mLT,优选采用口服给药。
本发明的方法和组合物想要在不成熟和成熟的脊椎动物中使用,特别是鸟类、哺乳动物和人。有用的抗原,做为例子但不做为限制,可包括来自细菌致病株抗原(酿脓链球菌、肺炎链球菌、淋病奈瑟氏球菌、脑膜炎奈瑟氏球菌、白喉棒状杆菌、肉毒梭状芽孢杆菌、产气荚膜梭状芽孢杆菌、破伤风梭状芽孢杆菌、流感嗜血杆菌、肺炎克雷伯氏杆菌、臭鼻克雷伯氏杆菌、鼻硬结克雷伯氏杆菌、金黄色葡萄球菌、霍乱弧菌、大肠杆菌、绿脓杆菌、胎儿弯曲菌(弧菌)、吸水性产气单孢菌、蜡样芽胞杆菌、缓慢爱德华氏菌、结肠炎耶乐森氏杆菌、鼠疫耶尔森氏杆菌、假结核耶尔森氏菌、痢疾志贺氏菌、福氏志贺氏菌、宋内志贺氏菌、鼠伤寒沙门氏杆菌、梅毒密螺旋体、极细密螺旋体、品他病密螺旋体、奋森氏疏螺旋体、包柔氏疏螺旋体菌、黄疸出血型钩端螺旋体、结核分枝杆菌、鼠弓形体、卡氏肺囊虫、土拉热弗郎西斯氏菌、流产布鲁氏菌、猪型布鲁氏菌、马尔他布鲁氏菌、支原体属、普氏立克次体、恙虫病立克次体、衣原体属;致病真菌(粗球孢子菌、烟曲菌、白色念珠菌、皮炎牙生菌、新型隐球菌、荚膜组织胞浆菌);原生动物(Entomoebahistolytica、tenas毛滴虫、人毛滴虫、阴道毛滴虫、刚比亚锥虫、罗德西亚锥虫、克氏锥虫、杜氏利什曼原虫、热带利什曼原虫、巴西利什曼锥虫、肺炎肺囊虫、间日疟原虫、恶性疟虫、三日疟原虫);或肠虫(蛲虫、毛首鞭虫、人蛔虫、旋毛线虫、粪类圆线虫、日本血吸虫、曼氏裂体吸虫、埃及血吸虫,和钩虫)以完整细胞形式或从技术上已知的设计用于生长所述生物体的培养基中部分分离的形式,或者通过基因工程技术或化学合成得到的所述生物体的保护抗原提呈给免疫系统。
其它相关的抗原为致病病毒(做为例子但不做为限制:痘病毒、疱疹病毒、单纯疱疹病毒1、单纯疱疹病毒2、腺病毒、乳多空病毒、肠病毒、小RNA病毒、细小病毒、呼肠病毒、逆转录病毒、流感病毒、副流感病毒、腮腺炎病毒、麻疹、呼吸合胞体病毒、风疹、虫媒病毒、弹状病毒、沙粒病毒、肝炎A病毒、肝炎B病毒、肝炎C病毒、肝炎E病毒、非A/非B肝炎病毒、鼻病毒、冠状病毒、轮状病毒和人免疫缺陷病毒)以完整的或从技术上已知的设计用于生长这些病毒的培养基中部分分离的形式,或者通过基因工程技术或化学合成得到的保护抗原提呈给免疫系统。
相关抗原的其它例子包括但不限于疫苗。这些疫苗的例子包括但不限于流感疫苗、百日咳疫苗、白喉和破伤风毒素与百日咳疫苗联用,肝
肝炎A疫苗、肝炎B疫苗、肝炎C疫苗、肝炎E疫苗、日本脑炎疫苗、疱疹疫苗、麻疹疫苗、风疹疫苗、腮腺炎疫苗、麻疹、腮腺炎和风疹的混合疫苗、乳头瘤病毒疫苗、细小病毒疫苗、呼吸合胞体病毒疫苗、Lyme病疫苗、脊髓灰质炎疫苗、疟疾疫苗、水痘疫苗、淋病疫苗、HIV疫苗、血吸虫病疫苗、轮状病毒(rota)疫苗、支原体疫苗、肺炎球菌疫苗、脑膜炎球菌疫苗、弯曲杆菌疫苗、霍乱疫苗、肠道病原性大肠杆菌疫苗、肠毒大肠杆菌疫苗等。这些可用已知的普通方法生产。总之,这些疫苗包含完整的生物体或用技术人员已知的技术培养和分离的病毒,或者包含通过基因工程技术或化学合成生产的这些生物体或病毒的相关抗原。其生产被解释但不限于如下:
流感疫苗:含有完整或部分血凝素、涎酸酶、核蛋白以及基质蛋白的疫苗,它们能通过用醚和去垢剂纯化接种于鸡胚的病毒,或通过基因工程技术或化学合成获得。
百日咳疫苗:含有完整或部分百日咳病毒、血凝素、以及K-凝集素的疫苗,它们能用甲醛从减毒毒素中获得,即通过盐析或超速离心从百日咳杆菌的培养基或菌体中提取,或通过基因工程技术或化学合成获得。
白喉和破伤风类毒素与百日咳疫苗联用:百日咳疫苗、白喉和破伤风类毒素的混合疫苗。
日本脑炎疫苗:含有完整或部分抗原蛋白的疫苗,它通过在小鼠脑内培养病毒,用离心或乙醇纯化病毒颗粒并灭活而获得,或通过基因工程技术或化学合成获得。
肝炎B疫苗:含有完整或部分通过盐析或超速离心分离并纯化HBs抗原获得的抗原蛋白的疫苗,它从带有肝炎的血液得到,或通过基因工程技术或化学合成获得。
麻疹疫苗:含有完整或部分生长在培养的鸡胚细胞或鸡胚中的病毒的疫苗,或者通过基因工程或化学合成得到的保护性抗原。
风疹疫苗:含有完整或部分生长在培养的鸡胚细胞或鸡胚中的病毒的疫苗,或者通过基因工程技术或化学合成得到的保护性抗原。
流行性腮腺炎疫苗:含有完整或部分生长在培养的兔细胞或鸡胚中的病毒的疫苗,或者通过基因工程或化学合成得到的保护性抗原。
麻疹、风疹和腮腺炎的混合疫苗:通过混合麻疹、风疹和腮腺炎疫苗得到的疫苗。
轮状病毒(Rota)疫苗:含有完整或部分生长于培养的MA 104细胞或从病人粪便分离的病毒的疫苗,或者通过基因工程或化学合成得到的保护抗原。
支原体的疫苗:含有完整或部分生长在支原体液体培养剂的支原体细胞,或者通过基因工程或化学合成得到的保护抗原的疫苗。
通过本方法可以获得有效预防的那些条件对技术人员是显而易见的。本发明的疫苗制剂组合物可以期望的比例通过将上述抗原和/或疫苗与mLT混合来制备。该制剂应该经严格无菌处理,并且每种成分也应无菌。热原或变应原自然应尽可能完全地除掉。本发明的抗原制剂可通过分别制备抗原本身和mLT使用。
而且本发明包含一个试剂盒,它含有有效量的抗原和佐剂有效量的mLT。在使用中该试剂盒的成分可以先混合起来再口服给药,或在一短的时间间隔内分别口服各成分。
本发明的疫苗制剂组合物可以液体或固体药物载体混合,该组合物可以片剂、胶囊、粉剂、粒剂、悬液或溶液形式,该组合物也可以含有适当的防腐剂、色素和香味剂或产生缓慢释放的试剂。本发明药物组合物制剂中可使用的可能载体包括但不限于明胶胶囊、蔗糖、纤维素衍生物如羧甲基纤维素钠、明胶、talc、硬脂酸镁、植物油如花生油等,甘油、山梨醇、琼脂和水。载体也可做为粘合剂以利于组合物的成片而方便口服。
本发明的疫苗制剂组合物可通过冷冻干燥或其它本领域技术人员已知的方法以一种稳定的储藏形式保存以随时使用。做为口服给药疫苗制剂可在缓冲盐水,牛奶,或其它任何生理上相容的液体介质中重配成悬液,该介质可以通过加入适当的色素和香味剂变的更易服用。
疫苗制剂组合物可在口服有效量胃酸中和剂后服用。虽然为此目的可使用许多化合物,但是优选碳酸氢钠。疫苗组合物或以肠溶包被胶囊(即只有通过胃后胶囊才溶解)递送。
6.实施例
下列实施例仅为解释而不是以任何方式限制本发明的范围而被介绍。
6.1 mLT的构建
野生型LT毒素被发现于能产生此毒素的产肠毒素的大肠杆菌株的天然存在质粒编码。本发明者先前已从叫做H 10407的大肠杆菌人分离物中克隆了LT基因。该亚克隆由插入pBR322质粒PstI位点的H 10407肠毒素质粒中的5.2kb DNA片段组成〔Clements et al.,1983,Infect.Immun.40:653〕。此叫做pDF82的重组质粒已被广泛鉴定并在天然LT启动子的控制下表达LT。此过程的下一步是将LT基因置于强启动子的控制之下,此处的pUC 18质粒的lac启动子。通过分别分离LT-A和LT-B的基因并将它们重组在载体质粒的一个盒中完成。这是一个重要步骤,因为它允许合理数量的LT的纯化并衍生为随后分析的突变体。该叫做pBD94的质粒图解见图1。
CT和LT都用连接A1和A2片段的胰蛋白酶敏感肽键合成。此肽键必须是缺口的,以使该分子“有毒”。对白喉毒素,原型A-B毒素和各种其它细菌毒素也一样。不论通过细菌蛋白酶或肠腔中的肠蛋白酶,即蛋白水解加工或激活,如果A1-A2键没有去掉,则A1片段不能达到其小肠上皮细胞基质层表面的靶上。与CT相比,LT当刚从细胞分离时不具完全的生物学活性。LT还需要蛋白水解成为完全活性的,而且蛋白水解激活不发生于细菌内部。所以,改变分子的毒性并不影响ADP-核糖基化酶活性的一种方法可能是通过基因操作,去掉连接A亚基的A1和A2成分的胰蛋白酶敏感氨基酸。如果该分子不能被蛋白水解切割,它就无毒。本领域的技术人员会预言该分子可能保持其ADP-核糖基化酶活性并最终保持其佐剂功能。
图1表示分开A1和A2片段的二硫包裹区域的序列。该区域中有一个精氨酸残基,它被认为是激活该分子毒性所必需的切割位点。该区域通过定位致突变被改变,以使分子对蛋白水解消化不敏感,最终无毒。
定位致突变通过将单链DNA与合成的寡核苷酸杂交完成的,该寡核苷酸除接近中央的失配区域外与单链模板互补。就是这个区域含有想要的一个或多个核苷酸改变。与单链靶DNA杂交后,该寡核苷酸用DNA聚合酶延伸以产生一个双链结构。缺口然后用DNA连接酶封口,针双链体结构转化到大肠杆菌宿主中。由于DNA的半保留复制模式,使用此方法的突变体理论产率是50%。实际上,产率更低。然而有些方法能改进产率并用于选择寡核苷酸定向突变体。采用的系统利用第二突变寡核苷酸,在两次突变策略中产生改变的限制性部位。
下一步是将Arg替换成另一氨基酸(即,GGA=甘氨酸替换AGA=精氨酸),虽然消除了蛋白水解位点但保留了阅读框。然后通过琼脂糖亲合层析,从已经测序证明的一个突变体(pBD95)纯化mLT。其它纯化方法对本领域的技术人员是显而易见的。此叫做LT(R192G)的突变LT通过SDS-聚丙烯酰胺凝胶电泳检测对胰蛋白酶敏感键的修饰。样品暴露或不暴露于胰蛋白酶进行检测并与天然(未修饰的)LT相比较。当与胰蛋白酶温育时不裂解为mLT A1和A2,因而表明去掉了对蛋白酶的敏感性。
6.2 mLT对Y-1肾上腺细胞的作用
本领域技术人员可能预言mLT在Y-1肾上腺细胞试验中没有活性。这个预言可能基于先前的发现〔Clements and Finkelstein,1979,Infect.Immun.24:760-769〕,即无缺口LT在此试验系统中比CT活性小1000倍以上,而且在该试验中胰蛋白酶处理激活的LT与CT具同等水平的生物学活性。此实验中无胰蛋白酶激活情况下观察到的LT残存活性被推测为不能被解释的一些残余蛋白酶活性的作用。例如,在Y-1肾上腺细胞传代培养过程中使用胰酶。因此推测不能被切口的LT在Y-1肾上腺细胞试验中可能完全无活性。结果见表1。
表I
毒素 | 被胰酶激活的 | 比活a |
霍乱毒素 | - | 15 |
LT | - | 60 |
LT | + | 15 |
LT(R192G) | - | 48,000 |
LT(R192G) | + | 48,000 |
a(>50%)细胞成为园形所需的最小剂量(每孔微微克) |
表I证明了出人意料的发现,即mLT即使没有被蛋白水解加工,在Y-1肾上腺细胞试验中仍保持活性的基本水平。如表I所示,经胰酶处理的CT和天然LT对Y-1肾上腺细胞具有相同水平的活性(15pg)。相比之下,mLT(48,000pg)比CT或天然LT活性小<1000倍,并不能被胰酶激活。残存的基础活性无疑反映了肾上腺细胞激活的不同的并到现在为止,除需要A1-A2连接的分离之外未知的通路。
6.3 mLT的ADP-核糖基化酶活性
因为用Gly192置换Arg192的突变不改变A1部分的酶促部位,所以本领域的技术人员可以预测mLT可能保留其ADP-核糖基化酶活性。为了检测该性质,采有NAD-Agmatine ADP-核糖转移酶试验,[Mosset al.,1993,J.Biol.Chem.268:6383-6387]。如图2所示,CT产生剂量依赖性ADP-ribosylagmatine水平的升高,该分子ADP-核糖转移酶活性的作用的升高。
表II
CT,天然CT和LT(R192G)的ADP-核糖转移酶活性 | |||||
实验 | 1 | 2 | 3 | 4 | 平均值±SEM |
无毒素 | ND | 9.12 | 5.63 | 14.17 | 9.64±2.48 |
1μg CT | ND | 17.81 | 17.60 | 25.75 | 20.39±2.68 |
10μg CT | ND | 107.32 | 111.28 | 104.04 | 107.55±2.09 |
100μg CT | 351.55 | 361.73 | 308.09 | ND | 340.46±16.45 |
100μg LT | 17.32 | 14.48 | 13.86 | ND | 15.22±1.07 |
100μg LT+胰酶 | 164.10 | 189.89 | 152.96 | ND | 168.98±10.94 |
100μgLT(R192G) | 14.58 | 12.34 | 9.30 | ND | 12.07±1.53 |
100μgLT(R192G)+Trypsin | 14.73 | 8.90 | 10.47 | ND | 11.37±1.74 |
ND=未做数据以fmoles min-1表示 |
表II以表格形式证明了出人意料的发现,即mLT不论有无胰酶激活,均缺少任何可察觉的ADP-核糖基化酶活性,即使没有改变酶促部位,并且在Y-1肾上腺细胞试验中有可证明的活性基础水平。
6.4 mLT的肠毒素活性
由于出人意料的发现即使酶促部位没有改变,在有或无胰酶激活条件下mLT缺乏任何可检测到的ADP-核糖基化酶活性,而且另外发现在Y-1肾上腺细胞试验中有基础活性水平,因此不清楚是否mLT会保留其肠毒性特性。mLT的理想制剂为保留其作为免疫佐剂的能力,但没有真正的或潜在的副作用,如与LT或CT的使用有关的腹泻。图3证明在伸展小鼠模型中,在125μg剂量下mLT不诱导网流体分泌。该剂量比在此模型中LT的佐剂有效剂量高出五倍多。重要的是,天然LT在此水平可看到其可能的毒性。
6.5 mLT的佐剂活性
由于mLT不具有可证明的ADP-核糖转移酶活性且非肠毒的,本领域的技术人员可能预测其缺乏佐剂性质。这个预测可能基于Lycke等的报道[Lycke et al.,1992,Eur.J.Immunol.22:2277-2281],在此人们清楚地知道:影响毒素ADP-核糖基化酶活性和改变增加细胞内CAMP水平的能力的变化也阻碍分子的佐剂功能。如上证明的,mLT无ADP-核糖基化酶活性,只在Y-1肾上腺细胞中有一些未确定的基础活性,mLT在伸展小鼠模型中不诱导网流体分泌。
为了检测mLT的佐剂活性,进行下列实验。免疫三组BALB/C小鼠。用钝头灌胃针给动物胃内接种(Popper & Sons,Inc.New HydePark,New York)。在0天,各组口服免疫如下:A组接受含5mgOVA的0.5ml PBS,B组接受含5mg OVA和25μg天然LT的0.5mlPBS,C组接受含5mg OVA和25μgmLT的0.5mlPBS。在7和14天再施用各剂量。在21天,所有动物腹腔注射含1μg OVA的20%Maalox进行加强免疫。i.p.后一周,处死接种的动物并用ELISA测试直接抗OVA和LT的血清IgG和粘膜IgA抗体。
用于ELISA的试剂和抗血清购自西格玛化学公司。用于ELISA的样品用磷酸盐缓冲盐水(PH7.2)-0.05%吐温20(PBS-TWEEN)进行系列稀释。对抗LT测定,预先用每孔1.5μg混合神经节苷脂(III型)包被微滴定板,然后每孔加1μg纯化的LT。抗OVA在先用每孔10μgOVA包被的微滴定板上测定。血清抗LT和抗OVA用偶联碱性磷酸酶兔抗鼠IgA(α-链特异的)抗血清测试后,再用偶联碱性磷酸酶的免抗羊IgG的抗血清测试。反应用3N NaOH终止。IgG和IgA的从纯化的鼠骨髓瘤蛋白的标准曲线确定(MOPC 315,gA(IgAl2)MOPC 21,gG1:Litton Bionetics,Inc.,Charleston,SC)。
6.5.1 血清IgG抗OVA
如图4A所示,口服OVA和LT的动物在随后用OVA(4,058μg/ml)胃肠外免疫后,比那些只灌注OVA并随后胃肠外用OVA免疫的(无可检测到的抗OVA反应)(Stadent t-检验P=.031)形成明显较高的血清IgG抗OVA反应。明显的,口服OVA和mLT的动物在随后用OVA(1,338μg/ml)胃肠外免疫后,也比那些只灌注OVA并随后胃肠外用OVA免疫的(无可检测到的抗OVA反应)(Student:t-检验P=.0007)形成明显较高的血清IgG抗OVA反应。
6.5.2 粘膜sIgA抗OVA
如图4B所示,在这些相同组的动物中在比较抗OVA IgA反应时获得相似的结论。口服OVA和LT的动物在随后用OVA(869nl/ml)胃肠外免疫后,比那些只灌注OVA并随后胃肠外用OVA免疫的(无可检测到的抗OVA反应)(Student t-检验p=.0131)形成明显较高的粘膜IgA抗OVA反应。如上,口服OVA和mLT的动物在随后用OVA(230ng/ml)胃肠外免疫后,比那些只灌注OVA并随后胃肠外用OVA免疫的(无可检测到的抗OVA反应)(Studentt-检验p=.0189)形成明显较高的粘膜IgA抗OVA反应。
6.5.3 血清IgG抗LT
在这些相同的动物中,还检测了LT和mLT引发抗LT抗体反应的能力。这是重要的,因为这将提供突变的LT除了做为其它蛋白的佐剂外,是否能防止诱导对其自身耐受的迹象。如图5A所示,口服OVA和LT的动物在随后用OVA(342μg/ml)胃肠外免疫后,比那些只灌注OVA并随后胃肠外用OVA免疫的(无可检测到的抗LT反应)(Studentt-检验p=.0005)形成明显较高的血清IgG抗LT反应。口服OVA和mLT的动物在随后用OVA(552μg/ml)胃肠外免疫后,比那些只灌注OVA并随后胃肠外用VOA免疫的(无可检测到的抗LT反应)(Studentt-检验p=.0026)也形成明显较高的血清IgG抗LT反应。
6.5.4 粘膜sIgA抗LT
如图5B所示,在这些相同组的动物中,在比较抗LT IgA反应时获得相似的结论。口服OVA和LT的动物在随后用OVA(4328ng/ml)胃肠外免疫后,比那些只灌注OVA并随后胃肠外用OVA免疫的(无可检测到的LT反应)(Student t-检验p=.0047)形成明显较高的粘膜IgA抗LT反应。如上,口服OVA和mLT的动物在随后用OVA(1463ng/ml)胃肠外免疫后,比那些只灌注OVA并随后胃肠外用OVA免疫的(无可检测到的抗LT反应)(Student t-检验p=.0323)也形成明显较高的粘膜IgA抗LT反应。
7.微生物的保藏
下列质粒于1994年8月18日保藏在美国典型培养物保藏中心(ATCC),Rockville,MD,并已被授予所示的保藏号:
质粒 保藏号
大肠杆菌LTR192G中的pBD95 ATCC 69683
在此描述并权利要求的发明不被在此公开的具体实施方案限制其范围,因为这些实施方案意欲作为本发明几个方面的解释。任何等效的实施方案意欲在本发明的范围内。实际上本发明的各种修改,除了那些在此已指出和描述的以外,对本领域的技术人员来说,会从前面的描述中变得明显。这些修改也被意欲处于附加的权利要求范围内。
还应该知道,所有的碱基对和氨基酸残基数目以及核苷酸和肽的大小是近似的,并为了描述而使用。
在此引用了许多文献,其全部公开内容在此全文引入做为参考。
国际申请号:PCT/US95/09005
PCT/RO/134表(1981年1月)
Claims (11)
1.一种突变体大肠杆菌热不稳定肠毒素全毒素,其中该全毒素A亚基的第192位氨基酸精氨酸被甘氨酸置换,该全毒素在Y-1肾上腺细胞测定中测得的毒性显著低于天然大肠杆菌热不稳定肠毒素全毒素,该全毒素具有免疫佐剂活性,但通过NAD-胍丁胺ADP-核糖基转移酶测定测得没有ADP-核糖基化酶活性。
2.权利要求1的突变体全毒素,它由质粒pBD95编码,该质粒可从ATCC保藏号为69683的大肠杆菌LTR192G获得,大肠杆菌LTR192G表达大肠杆菌热不稳定肠毒素的A亚基和B亚基。
3.一种疫苗制剂,该制剂含有一种与权利要求1的突变体全毒素组合的抗原。
4.权利要求3的疫苗制剂,该制剂还含有一种适当的载体、稀释剂或赋形剂。
5.权利要求3的疫苗制剂,其中抗原选自流感疫苗;水痘疫苗;白喉类毒素;破伤风类毒素;百日咳疫苗;日本脑炎疫苗;百日咳、白喉和破伤风类毒素的混合疫苗;莱姆病疫苗;脊髓灰质炎疫苗;疱疹疫苗;乳头状瘤病毒疫苗;乙型肝炎疫苗;轮状病毒疫苗;人免疫缺陷病毒;弯曲杆菌疫苗;霍乱疫苗;肠道病原性大肠杆菌疫苗;肠毒大肠杆菌疫苗;血吸虫病疫苗;麻疹疫苗;风疹疫苗;流行性腮腺炎疫苗;麻疹、风疹和腮腺炎联合疫苗;以及支原体疫苗。
6.一种用于在宿主中产生针对病原体的免疫反应的试剂盒,该试剂盒含有两种成分:(a)有效量的一种抗原;(b)佐剂有效量的突变体大肠杆菌热不稳定肠毒素全毒素,其中该全毒素A亚基的第192位氨基酸精氨酸被甘氨酸置换,该全毒素在Y-1肾上腺细胞测定中测得的毒性显著低于天然大肠杆菌热不稳定肠毒素全毒素,该全毒素具有免疫佐剂活性,但通过NAD-胍丁胺ADP-核糖基转移酶测定测得没有ADP-核糖基化酶活性,其中所述两种成分在口服可接受的载体中,并且所述成分可在混合到一起后给药或分别给药。
7.权利要求1的突变体金毒素在药物制备中的应用。
8.权利要求3的疫苗或权利要求6的试剂盒,其中所述抗原来源于细菌、病毒、原生动物、真菌、蠕虫和其它微生物病原体。
9.权利要求3的疫苗或权利要求6的试剂盒,其中所述抗原来源于一种肠毒细菌生物体。
10.权利要求9的疫苗或试剂盒,其中所述肠毒细菌生物体选自表达霍乱样毒素的肠毒细菌生物体。
11.权利要求10的疫苗或试剂盒,其中所述肠毒细菌生物体是大肠杆菌或霍乱弧菌。
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JP2849632B2 (ja) * | 1988-04-08 | 1999-01-20 | 社団法人北里研究所 | ワクチン製剤 |
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GB9326174D0 (en) * | 1993-12-22 | 1994-02-23 | Biocine Sclavo | Mucosal adjuvant |
US6019982A (en) * | 1994-08-26 | 2000-02-01 | The Administrators Of The Tulane Educational Fund | Mutant enterotoxin effective as a non-toxic oral adjuvant |
US6033673A (en) * | 1998-03-18 | 2000-03-07 | The Administrators Of Tulane Educational Fund | Double mutant enterotoxin for use as an adjuvant |
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