CN1241906A - 经皮免疫之佐剂 - Google Patents
经皮免疫之佐剂 Download PDFInfo
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- CN1241906A CN1241906A CN97180947A CN97180947A CN1241906A CN 1241906 A CN1241906 A CN 1241906A CN 97180947 A CN97180947 A CN 97180947A CN 97180947 A CN97180947 A CN 97180947A CN 1241906 A CN1241906 A CN 1241906A
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Abstract
经皮免疫体系不伤及皮肤而将抗原送递给免疫细胞,在动物或人中诱导免疫应答。对动物或人的完整皮肤经皮施用含有抗原和佐剂的制剂之后,该体系使用佐剂,优选使用ADP-核糖基化外毒素诱导抗原特异性免疫应答(如体液和/或细胞效应物)。在经皮送递体系中加入水合剂(如脂质体)、渗透增强剂或封闭性敷料可增强免疫效力。此体系可激活皮肤中的朗格汉斯细胞,将朗格汉斯细胞迁移至淋巴结并呈递抗原。
Description
发明背景
本发明涉及经皮免疫及可用于其中的佐剂,以诱导抗原特异性免疫应答。
经皮免疫既要求抗原能穿过通常无法穿透的皮肤表面屏障,又要求对该抗原的免疫应答。美国申请号08/749,164中用霍乱毒素作抗原,刺激产生了强烈的抗体应答,并有较好重复性;该抗原可以盐溶液形式用于皮肤,其中含或不含脂质体。本申请将展示使用佐剂的经皮免疫,所述佐剂例如有细菌外毒素及其亚基,以及相关毒素等。
Paul等(1995)曾报道使用转移体进行经皮免疫。在该报道中,利用转移体作为蛋白质(牛血清白蛋白和间隙连接蛋白)的载体,针对该蛋白质,产生了对抗原致敏化脂质体的补体介导的溶解作用。将含有该蛋白质的溶液施用于皮肤并未诱导免疫应答;只有转移体能运送抗原穿过皮肤,并产生免疫效应。如美国申请号08/749,164所述,转移体均非脂质体。
Paul等(1995)的图1显示,对抗原致敏的脂质体的裂解分析可知只有抗原加转移体的制剂才诱导免疫应答。溶液形式抗原的制剂、抗原与混合微团的制剂及抗原与脂质体的制剂(即smecticmesophases)用于皮肤时,所诱导的免疫效果均不及皮下注射的。因此,有阳性对照(抗原加转移体)可证实Paul等的阴性结论,即抗原加脂质体不能诱导经皮免疫。
Paul等(1995)在第3521页称,皮肤是有效的保护性屏障,“分子量多达750 DA的物质就无法穿过”,大免疫原无法透过完整皮肤实现非侵入性免疫。因此根据该文献,不应使用霍乱毒素(85,000道尔顿)这样的分子,因为不能指望它们穿过皮肤,就不能指望获得免疫效果。因此本发明公开之前的观点均认为皮肤屏障是佐剂或霍乱毒素类抗原无法穿透的。
Paul和Ceve(1995)在第145页称,“大分子通常不能透过哺乳动物皮肤。因此不可能用单纯肽或蛋白质溶液进行外表皮免疫。”他们得出结论,“皮肤上施用脂质体或混合微团式免疫原,不管它们是否与免疫佐剂脂质A联合作用,均如同单纯蛋白质溶液一样不具备生物学活性”。
Wang等(1996)将卵白蛋白(OVA)水溶液置于剃毛小鼠皮肤上以诱导过敏型应答,作为特应性皮炎模型。将小鼠麻醉,敷一封闭性补片,内含不超过10mg OVA,持续与皮肤接触4天。两周后重复操作一次。
在Wang等(1996)的图2中,用ELISA测定IgG2a抗体应答,结果显示没有对OVA的IgG2a抗体应答,但能测到与过敏反应相关的IgE抗体。进一步的实验中,小鼠被敷贴上更大面积OVA溶液,贴4天,隔两周重复一次,其重复5次,即小鼠共贴补片20天。如此高剂量的OVA仍然没能产生显著的IgG2a抗体,却产生了显著水平的IgE抗体。
作者们在第4079页称“我们建立了一种动物模型,显示外表皮接受蛋白质抗原(Ag)刺激,即使无佐剂,也能致敏动物,并诱导较强的Th2样应答,同时伴有高水平IgE”。外表皮大面积接触高剂量蛋白质抗原并不能产生显著的IgG抗体,但能诱导出IgE抗体,这是过敏型反应的标志。故Wang等(1996)告诫上述OVA接触方式是特应性皮炎的模型,并非免疫接种模式。因此,根据该文献的教导,可以推论,抗原经皮免疫方式如确有抗原穿过皮肤并诱导免疫应答,就会产生高水平IgE抗体。然而,我们意外地发现,将抗原盐溶液加佐剂置于皮肤上诱导的是高水平IgG和一些IgA,而非IgE。
与上述引用文献相比,本发明人发现,将抗原加佐剂施用于皮肤,产生了经皮送递抗原体系,可诱导IgG和IgA的抗原特异性应答。佐剂优选是ADP-核糖基化外毒素。此外,也可在经皮送递体系中使用水化作用、促穿透剂或封闭敷料。
发明简述
本发明旨在提供一种经皮免疫体系,在动物或人体内诱导免疫应答(如体液和/或细胞效应物)。
该体系只需简单地将含佐剂和抗原的制剂施用于生物体的完整皮肤,就能诱导针对该抗原的特异性免疫应答。
具体地说,该佐剂可激活免疫体系的抗原呈递细胞(如表皮朗格汉斯细胞,皮肤树突细胞,树突细胞,巨噬细胞,B淋巴细胞),还/或可诱导抗原呈递细胞吞噬抗原。然后抗原呈递细胞将抗原呈递给T细胞和B细胞。在朗格汉斯细胞的情形中,抗原呈递细胞可能从皮肤移行至淋巴结,将抗原呈递给淋巴细胞(如T和/或B细胞),由此诱导抗原特异性免疫应答。
除了激发免疫反应导致产生抗原特异性B淋巴细胞和/或T淋巴细胞(包括细胞毒性T淋巴细胞,CTL)之外,本发明的另一目的是用经皮免疫体系影响抗原特异性T辅助淋巴细胞(Th1,Th2或两种全影响),从而对免疫体系的各成分进行正和/或负调节。
在本发明的第一个实施方案中,将含有佐剂和抗原的制剂施用于生物体的完整皮肤后,抗原被呈递给免疫细胞,未经皮肤穿孔就诱导产生了抗原特异性免疫应答。该制剂中可以包含其他抗原,使得经皮施用制剂可诱导对多种抗原的免疫应答。此时,所用各种抗原可以是来源相同,也可不同,但应具备不同化学结构,以便诱导对不同抗原特异的免疫应答。抗原特异性淋巴细胞有可能参与免疫应答,如在B淋巴细胞的参与下,抗原特异性抗体可能是一部分免疫应答。
本发明的第二个实施方案中,上述方法被用来处理生物体。如果所用抗原来自病原体,则进行接种,使该生物体可抵抗该病原体的侵袭,或抵抗其致病作用,如因毒素分泌而导致的致病作用。包含肿瘤抗原的制剂也许能治疗癌症,包含自身抗原的制剂也许能治疗该生物体自身免疫体系所致疾病(如自身免疫疾病),包含变应原的制剂也许可用于对变态反应性疾病作免疫治疗。
在本发明的第三个实施方案中,提供了可用于上述方法的补片。补片中含有敷料,有效量的抗原和佐剂。敷料可为封闭性或非封闭性的。补片中可能还包含其他抗原,使得敷用该补片后,会诱导针对多种抗原的免疫应答。在这种情况下,所用抗原可以来源相同,也可不同,但应有不同化学结构,以便激发对不同抗原特异的免疫应答。为达到有效处理效果,可间隔性地多次重复敷用多块补片一段时间,也可持续敷用一段时间。
此外,在本发明的第四个实施方案中,使用一块或多块补片,将制剂敷到完整皮肤上,接触部位涵盖不止一个引流淋巴结区。制剂中可能含有其他抗原,使得敷到完整皮肤上时,可诱导对多种抗原的免疫应答。在这种情况下,所用抗原可来源相同,也可不同,但应有不同化学结构,以便诱导对诸不同抗原特异的免疫应答。
本发明的产品及方法可用于治疗已有疾疾,预防疾病,或减轻病性和/或缩短病程。但以不诱导过敏反应、特应性疾病、皮炎或接触性超敏反应为佳。
制剂中不止含有抗原和佐剂,还可含水化剂(如脂质体),促穿透剂,或二者均有。例如,抗原-佐剂制剂中可能还含有用AQUAPHOR制成的乳剂(凡士林,矿物油,矿物蜡,羊毛蜡,泛酰醇,bisabol和甘油),乳剂(如水性乳膏),水包油乳剂(如油性乳膏),无水脂质加水包油乳剂,无水脂质加油包水乳剂,脂肪,蜡,油,蛙酮,湿润物(如甘油),凝胶(如SURGILUBE,KY凝胶),或以上各试剂的组合物。制剂可以制成水溶液形式。
制剂中较优选不含有机溶剂。可在用酒精擦拭皮肤后施用制剂。但不优选在经脱发剂处理除去了角质细胞层的皮肤表面给药。
抗原可得自能感染生物体的病原体(如细菌、病毒、真菌或寄生虫),或可得自细胞(如肿瘤细胞或正常细胞)。抗原也可以是肿瘤抗原或自身抗原。从化学角度看,抗原可以是糖类、糖脂、糖蛋白、脂类、脂蛋白、磷脂、多肽或以上各类的化学或重组偶联物。抗原分子量可以大于500道尔顿,较优选大于800道尔顿,更优选大于1000道尔顿。
抗原可经重组方式、化学合成方式获得,也可从天然来源中纯化而得。较优选蛋白性抗原或与多糖的偶联物。抗原可以是至少部分纯化成无细胞形式。或者,抗原可以是活病毒、减毒活病毒或灭活病毒的形式。
佐剂的引入可能对免疫应答产生增强或调节作用。而且,选择适当的抗原或佐剂可优先诱导体液免疫或细胞免疫应答,产生特异性抗体同种型(如IgM,IgD,IgA1,IgA2,IgE,IgG1,IgG2,IgG3,IgG4,或其组合),和/或产生特异性T细胞亚群(如CTL,Th1,Th2,TDTH,或其组合)。
佐剂较优选为ADP-核糖基化外毒素或其亚基,或者也可用朗格汉斯细胞的激活剂。
另一选择是,抗原、佐剂或二者可作为编码相应抗原或佐剂的核酸形式(如DNA,RNA,cDNA,cRNA)加入到制剂中。该技术称基因免疫接种。
本发明中术语“抗原”指被呈递给生物体的免疫细胞时,能诱导特异性免疫应答的物质。抗原可包含能被B细胞受体(即B细胞的细胞膜上抗体)或T细胞受体识别的一个或多个免疫原性表位。一种分子可能既是抗原又是佐剂(如霍乱毒素),因此,制剂中可能只含一种成分。
本发明所用术语“佐剂”指加入制剂中以辅助诱导对抗原之免疫应答的物质。一种物质可能既是佐剂又是抗原,可诱导免疫刺激反应,及特异性抗体或T细胞应答。
本发明中术语“有效量”指能诱导抗原特异性免疫应答的抗原量。这种免疫应答的诱导可用于治疗中,比如免疫保护、脱敏、免疫抑制、自身免疫疾病调节、癌症免疫监视的强化或对已患的传染病进行的治疗接种。
本发明中术语“引流淋巴结区”指淋巴汇集后滤过一组淋巴结(如颈部、腋窝、腹股沟、肱骨内上髁(epitrochelear)及胸、腹部的淋巴结)所流经的解剖学区域。
附图简述
图1显示霍乱毒素(CT)诱导增强主要组织相容性复合体(MHC)II类抗原在朗格汉斯细胞(LC)表面的表达,诱导LC的形态改变,及诱导LC的丢失(推测是通过迁移)。对BALB/C小鼠(H-2d)于耳部经皮免疫含250μg CT或其B亚基(CTB)的盐溶液。已有事前实验证实小鼠耳部皮肤易于被免疫(单次免疫接种后可产生7000ELISA单位的抗CT抗体)。16小时后,制备表皮片,并对MHC II类分子染色(标度尺50μm)。各部分指:(A)仅用盐溶液作阴性对照,(B)用CT盐溶液进行的经皮免疫,(C)用CTB盐溶液进行的经皮免疫,(D)皮内注射肿瘤坏死因子α(10μg)作阳性对照。
发明详述
经皮免疫体系将物质送递至能产生免疫应答的特殊类群细胞(如抗原呈递细胞,淋巴细胞)(Bos,1997)。这类物质被称为抗原。抗原可由以下化学物质组成,如糖类、糖脂、糖蛋白、脂类、脂蛋白、磷脂、多肽、蛋白质,及以上各类的偶联物,或已知能诱导免疫应答的其他任何物质。抗原可以是完整生物体,如细菌或病毒颗粒;抗原也可得自完整细胞或膜的提取物或裂解产物;或者,抗原可经化学合成,或重组产生,或通过灭活病毒得到。
制备药物制剂的方法为本领域熟知,借此方法,将抗原和佐剂与药学可接受的载体结合使用。合适的载体及其制备已有文献描述,如E.W.Martin的Remington′s药物科学。这类制剂应含有效量的抗原和佐剂,及适当量的载体,以制备药学可接受的组合物,使之适于对人或动物施用。制剂可以乳膏、乳剂、凝胶、洗剂、软膏、糊剂、溶液、悬浮液形式或本领域已知的其他形式施用。尤其优选能增强皮肤水化作用、穿透作用或同时有这两类作用的制剂。其中还可掺入其他药学可接受的添加剂,包括如稀释剂、粘合剂、稳定剂、防腐剂和着色剂。
增加对角质层的水化可提高所用溶液的经皮吸收速率(Roberts和Walker,1993)。本发明的“促穿透剂”不包括以下物质,如:水,生理缓冲液,盐溶液,及无法穿透皮肤的酒精。
本发明的目的之一是提供一种通过完整皮肤的新免疫接种方法,其中毋需穿透皮肤。该经皮免疫体系提供了一种方法,使抗原和佐剂可被运送至免疫体系,特别是送至皮下的特化抗原呈递细胞,如朗格汉斯细胞。
没有任何特别的理论支持,只是为了解释我们的发现,我们假定该经皮免疫送递体系携带抗原至免疫体系的细胞,在此诱导免疫应答。抗原可能穿过皮肤的正常保持性外表层(即角质层),直接诱导免疫应答,或通过某种抗原呈递细胞(如巨噬细胞、组织巨噬细胞、朗格汉斯细胞、树突细胞、皮肤树突细胞、B淋巴细胞或枯否细胞),将加工后的抗原呈递给T淋巴细胞,从而诱导免疫应答。也可能抗原借助毛囊或皮肤小器官(如汗腺、脂腺)穿过角质层。
用细菌ADP-核糖基化外毒素(bARE)进行的经皮免疫可能靶向的是上皮朗格汉斯细胞,它是最有效的抗原呈递细胞(APC)之一(Udey,1997)。我们发现,将bARE的盐溶液施用于皮肤外表面可激活朗格汉斯细胞。朗格汉斯细胞指导特异性免疫应答,即朗格汉斯细胞通过吞噬抗原、迁移至淋巴结、并在其中充当APC,将抗原呈递给淋巴细胞(Udey,1997),从而诱导有效的抗体应答。尽管皮肤通常被认为是生物入侵的屏障,但遍布表皮的大量朗格汉斯细胞证实了这道屏障有缺陷,这些细胞的存在意义就在于当有生物体从皮肤入侵时,即产生对抗该生物体的免疫应答(Udey,1997)。
Udey(1997)的观点是:
“朗格汉斯细胞来源于骨髓,它们存在于所有哺乳类的复层扁平上皮中。这些细胞构成了未发炎表皮中存在的所有辅助细胞活性,在当前实例中,它们是启动和扩大针对外表皮所施抗原产生的免疫应答所必需的。朗格汉斯细胞是有效辅助细胞家族(‘树突细胞’)的成员,这类细胞广泛分布于上皮和实体器官及淋巴样组织中,但很少被提及…”
“现已认识到朗格汉斯细胞(可能还有其他树突细胞)的生命周期至少有两个截然不同的时期。表皮中的朗格汉斯细胞组成了捕获抗原的监视细胞常规网络。表皮朗格汉斯细胞能摄入颗粒,包括微生物,并有效地加工处理复合抗原。但它们只表达低水平的MHC I类和II类抗原及共同刺激分子(ICAM-1,B7-1和B7-2),也难以刺激未接触抗原的T细胞。与抗原接触后,一些朗格汉斯细胞被激活,从表皮移出,并迁移至局部淋巴结的T细胞依赖区,在此处作为成熟树突细胞定居下来。在移出表皮并迁移至淋巴结的过程中,携带抗原的表皮朗格汉斯细胞(现在是“信使”)在形态、表面表型和功能方面发生巨大变化。与表皮朗格汉斯细胞相比,淋巴样树突细胞基本上没有吞噬能力,也不能有效地加工蛋白质抗原,但可表达高水平的MHC I类和II类抗原及各类共同刺激分子,而且是已鉴定出的原初T细胞的最强有力刺激物。”
我们预计,可以利用表皮朗格汉斯细胞的有效抗原呈递能力,开发经皮送递疫苗。应用皮肤免疫体系产生经皮免疫应答要求经被动扩散只将疫苗抗原送递至角质层(由角质化细胞和脂质共同组成的皮肤最外层)朗格汉斯细胞,及随后激活朗格汉斯细胞,使之摄取抗原,迁移至B细胞滤泡和/或T细胞依赖区,并将抗原呈递给B细胞和/或T细胞(Stingl等人,1989)。如果待被朗格汉斯细胞吞噬的抗原(如BSA)不是bARE,则这些抗原也可被带到淋巴结,并呈递给T细胞,随后诱导针对该抗原(如BSA)的特异性免疫应答。故经皮免疫的特征之一是对朗格汉斯细胞的激活,推测这种激活是由细菌ADP-核糖基化外毒素、ADP-核糖基化外毒素结合性亚基(如霍乱毒素B亚基)或其他朗格汉斯细胞激活物进行的。
经由朗格汉斯细胞活化、迁移和呈递抗原进行的经皮免疫机制已被以下证据证实:在用CT或CTB经皮免疫的表皮小片中,表皮朗格汉斯细胞增加了对MHC II类的表达。此外,由经皮免疫诱导的抗体应答水平和同种型变换成IgG占优势,通常是由抗原呈递细胞如朗格汉斯细胞或树突细胞(Janeway和Travers,1996)刺激的T辅助细胞以及由IgG1和IgG2a的产生揭示的Th1和Th2两条途径的激活(Paul和Seder,1994;Seder和Paul,1994)所造成的。而小鼠经CT+OVA免疫后显示有针对抗原OVA的T细胞增殖。另一方面,用直接激活B细胞的胸腺非依赖性抗原1型(TI-1)可诱导产生巨大的抗体应答(Janeway和Travers,1996)。
接触性皮炎和特应性皮炎可代表更常见的一类皮肤免疫应答。接触性皮炎是朗格汉斯细胞激活的致病性表现,它是由朗格汉斯细胞所导致的。朗格汉斯细胞吞噬抗原,迁移至淋巴结,呈递抗原,使T细胞致敏从而在皮肤感染处产生强烈的破坏性细胞应答(Dah1,1996;Leung,1997)。特应性皮炎中也发生类似的朗格汉斯细胞作用,但与Th2细胞有关,并通常伴随产生高水平的IgE抗体(Dah1,1996;Leung,1997)。
另一方面,用霍乱毒素及相关bARE进行经皮免疫是一种新型免疫应答,在霍乱毒素免疫接种后的24,48和120小时内并未发生淋巴细胞浸润,表明没有出现表面的和微观的免疫接种后皮肤变化(即无发炎皮肤)。这说明朗格汉斯细胞“含有未发炎表皮中存在的所有辅助细胞活性,在当前实例中,是启动和扩大针对外表皮所施抗原的免疫应答所必需的”(Udey,1997)。此处经皮免疫应答的独特性还体现在抗原特异性IgG抗体的高水平、所产生的抗体类别(如IgM,IgG1,IgG2a,IgG2b,IgG3和IgA),以及无抗CT的IgE抗体。
故我们发现,细菌衍生毒素用于皮肤表面时,可激活朗格汉斯细胞或其他抗原呈递细胞,并诱导有效的免疫应答,表现为产生高水平的抗原抗异性循环IgG抗体。对于一些置于皮肤上时本身并无免疫原性的蛋白质,可在经皮免疫中使用这种佐剂,以增加对所述蛋白质的IgG抗体应答。
经皮靶向朗格汉斯细胞还可用于使其抗原呈递功能失活,从而阻止发生免疫或致敏作用。使朗格汉斯细胞失活的技术包括,如用白细胞介素10(Peguet-Navarro等,1995),白细胞介素1β的单克隆抗体(Enk等,1993),或经超抗原消除朗格汉斯细胞,如经葡萄球菌肠毒素A(SEA)诱导表皮朗格汉斯细胞缺失(Shankar等,1996)。
经皮免疫可由CT、LT或诸如CTB这样的亚基的神经节苷脂GM1结合活性诱导(Craig和Cuatrecasas,1975)。神经节苷脂GM1是广泛存在于所有哺乳动物细胞膜上的糖脂(Plotkin和Mortimer,1994)。CT B亚基五聚体结合至细胞表面时,会形成亲水孔,可容许A亚基穿过脂质双层(Ribi等,1988)。
我们已显示,用CT或CTB进行经皮免疫,可能需神经节苷脂GM1结合活性。当用CT、CTA和CTB经皮免疫小鼠时,只有CT和CTB引起了免疫应答。CTA含有ADP-核糖基化外毒素活性,但只有含结合活性的CT和CTB才能诱导免疫应答,说明经皮肤免疫必需B亚基,且只要B亚基即足够了。我们得出结论,朗格汉斯细胞或其他抗原呈递细胞可由于CTB结合至它的表面而被激活。抗原
本发明的抗原可经重组表达,较优选是作为与亲和标记或表位标记的融合体表达(Summers和Smith,1987;Goeddel,1990;Ausubel等,1996);化学合成的寡肽,不管是游离的还是偶联于载体蛋白上,均可用于获得本发明的抗原(Bodanszky,1993;Wisdom,1994)。寡肽被视为多肽的一类。
较优选长为6至20个残基的寡肽。多肽也可被合成为分支结构,就如美国专利号5,229,490和5,390,111所述的那些多肽。抗原性多肽包括,如合成或重组B细胞和T细胞表位,T细胞通用表位,以及来自不同生物体或疾病的B细胞表位和T细胞表位的混合体。
经重组或肽合成方式获得的抗原,及得自天然来源或提取物中的本发明抗原,均可借助于抗原的物理或化学性质纯化,较优选通过分级分离或层析进行纯化(Janson和Ryden,1989;Deutscher,1990;Scopes,1993)。
可以用多价抗原制剂同时诱导对不止一种抗原的免疫应答。偶联物可用于诱导对多种抗原的免疫应答,或可用于加强免疫应答,或用于二者。此外,毒素免疫可用类毒素加强,或类毒素免疫可用毒素加强。经皮免疫可用于最初经其他免疫途径如注射、口服或经鼻途径诱导的应答进行加强。
抗原包括如毒素、类毒素、其亚基,或其组合(如霍乱毒素、破伤风类毒素)。
可将抗原溶于水或其他溶剂(如甲醇)中,或溶于缓冲液中。适合的缓冲液有,但不限于,不含Ca++/Mg++离子的磷酸缓冲盐溶液(PBS),生理盐水(溶于水中的150mM NaCl),及Tris缓冲液。可以将不溶于中性缓冲液中的抗原溶于10mM醋酸中,再用中性缓冲液(如PBS)稀释至所需体积。至于只在酸性pH可溶的抗原,可用稀醋酸溶解,再用酸性pH的醋酸盐-PBS作稀释剂。本发明中甘油可作为合适的非水性缓冲液使用。
如果抗原(如甲肝病毒)本身并不可溶,则抗原可以悬浮液或甚至是凝聚物的形式存在于制剂中。
疏水性抗原可溶于去污剂中,如含跨膜结构域的多肽抗原。而且,对于含脂质体的制剂,可以将去污剂溶液中的抗原(如细胞膜的提取物)与脂类混合,再经稀释、透析或柱层折去除去污剂即可形成脂质体。某些抗原,如来自病毒(如甲肝病毒)的那些抗原,并不需要它们本身可溶,而可以病毒体形式直接掺入到脂质体中(Morein和Simons,1985)。
Plotkin和Mortimer(1994)提供的抗原可用于免疫接种动物或人体,以诱导对具体病原体特异的免疫应答,他们还提供了制备抗原、确定抗原的合适剂量、免疫应答诱导量的测定以及治疗病原体(如细菌、病毒、真菌或寄生虫)所致感染的方法。
细菌包括例如:炭疽,弯曲杆菌,霍乱菌,白喉菌,肠产毒性大肠杆菌,贾第鞭毛虫,淋球菌,幽门螺杆菌(Lee和Chen,1994),B型流感嗜血杆菌,非典型流感嗜血杆菌,脑膜炎球菌,百日咳菌,肺炎球菌,沙门氏菌,志贺氏菌,B族链球菌,A族链球菌,破伤风菌,霍乱弧菌,耶尔森氏菌,葡萄球菌,各种假单胞菌及各种梭菌。
病毒包括:腺病毒,登革病毒1至4血清型(Delenda等,1994;Fonseca等,1994;Smucny等,1995),埃博拉病毒(Jahrling等,1996),肠道病毒,甲、乙、丙、丁、戊各血清型肝炎病毒(Blum,1995;Katkov,1996;Lieberman和Greenberg,1996;Mast,1996;Shafara等,1995;Smedila等,1994;美国专利号5,314,808和5,436,126),单纯疱疹病毒1型或2型,人类免疫缺陷病毒(Deprez等,1996),流感病毒,日本马脑炎病毒,麻疹病毒,诺沃克病毒,乳头瘤病毒,细小病毒B19,脊髓灰质炎病毒,狂犬病毒,轮状病毒,风疹病毒,牛痘病毒,含有可编码其他抗原(如疟疾抗原)的基因的牛痘病毒构建物,水痘病毒以及黄热病毒。
寄生虫包括:溶组织内阿米巴(Zhang等,1995);疟原虫(Bathurst等,1993;Chang等,1989,1992,1994;Fries等,1992a,1992b;Herrington等,1991;Khusmith等,1991;Malik等,1991;Migliorini等,1993;Pessi等,1991;Tam,1988;Vreden等,1991;White等,1993;Wiesmueller等,1991),利什曼原虫(Frankenburg等1996),弓形体和蠕虫。
抗原也包括用于生物战的那些物质,如蓖麻蛋白,对其可通过抗体达到保护效果。佐剂
制剂中还含有佐剂,虽然单单一种分子可能既有佐剂性质又有抗原性质(如霍乱毒素)(Elson和Dertzbaugh,1994)。佐剂是用于特异性或非特异性地增强抗原特异性免疫应答的物质。通常在呈递抗原之前混合佐剂和制剂,但也可以在一个较短的时间间隔内分别呈递抗原和佐剂。
佐剂包括例如油乳剂(如完全或不完全弗氏佐剂),趋化因子(如防卫素1或2,RANTES,MIP1-α,MIP-2,白细胞介素8)或细胞因子(如白细胞介素-1β、-2、-6、-10或-12;γ-干扰素;肿瘤坏死因子-α;或粒细胞-单核细胞集落刺激因子(Nohria和Rubin的综述,1994),胞壁酰二肽衍生物(如murabutide,苏氨酰基-MDP或胞壁酰三肽),热休克蛋白或其衍生物,Leishmaniamajor LeIF衍生物(Skeiky等1995),霍乱毒素或霍乱毒素B,脂多糖(LPS)衍生物(如脂质A或单磷酰基脂质A),或超抗原(Saloga等,1996)。可用于免疫接种的佐剂亦可参见Richards等(1995)。
所选佐剂可优选诱导抗体或细胞效应物,特异性抗体同种型(如,IgM,IgD,IgA1,IgA2,分泌型IgA,IgE,IgG1,IgG2,IgG3和/或IgG4),或特异性T细胞亚群(如CTL,Th1,Th2和/或TDTH)(Glenn等,1995)。
霍乱毒素是细菌外毒素,属ADP-核糖基化外毒素(bARE)家族。大多数bARE均为A∶B二聚体形式,有一个结合性B亚基和一个含ADP-核糖基转移酶的A亚基。这类毒素包括白喉毒素,假单胞菌外毒素A,霍乱毒素(CT),大肠杆菌不耐热肠毒素(LT),百日咳毒素,肉毒梭菌毒素C2,肉毒梭菌毒素C3,泥渣梭菌胞外酶,蜡状芽孢杆菌胞外酶,假单胞菌外毒素S,金黄色葡萄球菌EDIN和球形芽孢杆菌毒素。
霍乱毒素是由A和B亚基组成的范例bARE。B亚基是结合亚基,由B亚基五聚体组成,其非共价结合于A亚基。B亚基五聚体排列成对称的环形结构,与靶细胞表面的GM1神经节苷脂结合。A亚基使包括Gs蛋白在内的异三聚体GTP蛋白(G蛋白)亚群的α亚基发生ADP核糖基化,从而提高胞内环腺苷酸(cAMP)水平。就霍乱而言,这将刺激肠细胞释放离子和液体。
霍乱毒素(CT)及其B亚基(CTB)用作肌内或口服免疫原时具有佐剂性质(Elson和Dertzbaugh,1994;Trach等,1997)。另一种抗原-大肠杆菌不耐热肠毒素(LT),与CT在氨基酸水平有80%同源,并具有相似的结合特性;它也可在肠内结合GM1神经节苷脂受体,并有相似的ADP-核糖基化外毒素活性。另一种bARE,假单胞菌外毒素A(ETA),能结合α2巨球蛋白受体-低密度脂蛋白受体相关蛋白(Kounnas等,1992)。bARE由Krueger和Barbieri(1995)进行了综述。
口服、鼻内和肌内途径施用CT带来的毒性限制了它可用作佐剂的剂量。在一项对比试验中,肌内注射CT后,在注射部位引发了大面积肿大。相比之下,等量或更大剂量的CT用于皮肤上则无毒性产生。
以下实施例显示,霍乱毒素(CT)、其B亚基(CTB)、大肠杆菌不耐热肠毒素(LT)及百日咳毒素均是经皮免疫的有效佐剂,可诱导高水平的IgG抗体,但不产生IgE抗体。其中还显示,没有CT只有CTB也能诱导产生高水平IgG抗体。所以说bARE及其衍生物以简单的溶液形式用于皮肤外表面时均能有效引起免疫。此外,这些实施例还证实CT、CTB和bARE既可作佐剂,又可作抗原。
BSA用于皮肤上时一般并没有免疫原性,但与佐剂如CT混合后,可诱导抗BSA抗体。单独用白喉类毒素不能诱导免疫应答,但同时用百日咳毒素作佐剂就可以诱导产生对白喉类毒素的免疫应答。所以说,bARE在经皮免疫体系中可用作非免疫原性蛋白质的佐剂。
其它蛋白质也可以既作佐剂又作抗原。如FLUZONE(Lederle),分离的流感病毒甲型和乙型病毒体疫苗中含有强免疫免原性神经氨酸酶和血凝素,可作为其自身抗原和佐剂,经皮引起有效免疫,产生保护作用。类毒素,如经甲醛处理形成的白喉类毒素,经过氧化氢处理形成的百日咳类毒素;或是毒素突变体,如经基因工程技术破坏核糖基转移酶活性而被类毒素化的霍乱毒素或大肠杆菌不耐热肠毒素,仍可能保留了佐剂特性,既可用作抗原,又可用作佐剂。
对白喉、百日咳和破伤风(DPT)等危及生命的感染可经诱导高水平的循环性抗毒素抗体而对机体产生保护作用。有些研究人员认为百日咳是个例外,其保护作用必需有针对该侵染生物体其它部位的抗体,尽管对此有争议(见Schneerson等,1996),且最新代的非细胞性百日咳疫苗仍以PT(百日咳毒素)作为其中的成分(Krueger和Barbieri,1995)。DPT致病的病理机制直接与其毒素的作用有关,抗毒素抗体在保护作用中当然占有重要位置(Schneerson等,1996)。
一般而言,毒素可经化学失活处理使其毒性减弱但仍保留免疫原性,成为类毒素。我们预计,在经皮免疫体系中用基于毒素的免疫原和佐剂能获得有足够保护作用的抗毒素水平以抵抗这些疾病。抗毒素抗体可通过用毒素或基因工程减毒的类毒素本身、或联合使用类毒素与佐剂如CT、或单独用类毒素而免疫产生。预计经基因工程改造后,其ADP-核糖基化外毒素活性有所改变,但结合活性未变的类毒素化毒素,在用于经皮免疫时,可以作为抗原呈递细胞的无毒性激活物,发挥特别作用。
我们预计CT可作为佐剂,通过经皮免疫诱导抗原特异性CTL(见Bowen等,1994;CT在口服免疫接种中用作佐剂的用途参见Porgador等,1997。)
bARE佐剂可经化学作用偶联至其他抗原,如糖类、多肽、糖脂和糖蛋白抗原。这些抗原与毒素、其亚基或类毒素的化学偶联物在施用于外表皮时预计能增强对这些抗原的免疫应答。
为克服毒素的毒性问题(例如,已知白喉毒素很毒,其一个分子即可杀死一个细胞),并克服处理这种强毒性毒素如破伤风毒素时的危险性,一些工作者采用了重组方法生产基因工程化类毒素。这依据的是通过基因缺失使ADP-核糖基转移酶的催化活性失活。这些毒素与天然毒素相比,保留了结合能力,但无毒性。该方法由Burnette等,(1994),Rappuoli等(1995)和Rappuoli等(1996)进行了描述。这种基因工程类毒素化的外毒素可用于经皮免疫体系中,这是因为类毒素被视为没有毒性,也就不会带来安全问题。它们既可为抗原,又可作佐剂,增强对其自身或所添加抗原的免疫应答。另有好几种技术可用于对会产生同样问题的毒素进行化学类毒素化(Schneerson等,1996)。或者,可利用毒素或类毒素的片段,如破伤风毒素的C片段。在某些应用中,尤其在幼儿应用中,由于摄入毒素(如白喉毒素)可能会引起不利反应,这些技术就显得很重要。
或者,可以利用朗格汉斯细胞激活物作佐剂。这种激活物的例子包括:热休克蛋白诱导物;接触致敏剂(如三硝基氯苯,二硝基氟苯,氮芥,十五烷基儿茶酚);毒素(如志贺菌毒素,葡萄球菌肠毒素B);脂多糖,脂质A及其衍生物;细菌DNA(Stacey等,1996);细胞因子(如肿瘤坏死因子-α,白细胞介素-1β,-10,-12);以及趋化因子(如防卫素1或2,RANTES,MIP-1α,MIP-2,白细胞介素8)。
本发明中可以使用不同佐剂的组合物。例如,含CpG核苷酸序列的细菌DNA与ADP-核糖基化外毒素的组合物可以用于介导对经皮施用之抗原的T辅助细胞应答。因此,利用非甲基化CpG细菌DNA,或其他蛋白质,如LeIF或钙通道阻断剂,可使针对佐剂为CT的抗原的Th1或Th2样应答发生切换。
CpG结构模式可使免疫系统识别其致病性部位,激发固有免疫应答,产生适应性免疫应答(Medzhitov和Janeway,Curr.Opin.Immunol.,1997年,第9期,4-9页)。这些结构被称为病原体相关性分子模式(PAMP),包括脂多糖、磷壁酸、非甲基化CpG基元、双链RNA和mannin。
PAMP诱导可介导炎症反应的内源信号,作为T细胞功能的共同刺激物,并控制效应物的功能。PAMP诱导这些反应的能力对于它们作为佐剂的能力是很重要的,其靶点是APC,如巨噬细胞和树突细胞。皮肤的抗原呈递细胞亦可由穿过皮肤送递的PAMP所刺激。如朗格汉斯细胞这类树突细胞,可被皮肤上的PAMP溶液与经皮弱免疫原性分子所激活,并被诱导迁移,将该弱免疫原性分子呈递给淋巴结中的T细胞,诱导针对该弱免疫原性分子的抗体应答。PAMP亦可与其他皮肤佐剂(如霍乱毒素)结合使用,以诱导不同的共同刺激分子,并控制不同效应物功能,从而指导免疫应答,如从Th2至Th1的应答。
若免疫抗原有足够能力激活朗格汉斯细胞,则不必另用佐剂,如既可作抗原又可作佐剂的CT就是如此。预计全细胞制剂、活病毒、减毒病毒、DNA质粒和细菌DNA均能有效地经皮免疫。或许可以用低浓度的接触致敏剂或其他朗格汉斯细胞激活物诱导免疫应答,而不会带来皮肤损伤。脂质体及其制备
脂质体为封闭小泡,内包水相空间。内部区室与外界介质间被由分散的脂质分子组成的脂质双层所隔开。本发明中,可穿过完整皮肤将抗原送递至免疫体系的特化细胞,在那里诱导抗原特异性免疫应答。经皮免疫可用脂质体完成;但正如实施例中所示,脂质体并非激发抗原特异性免疫应答所必需的。
可采用多种技术,利用膜脂质制备脂质体(由Gregoriadis,1993综述)。可先形成脂质体,再混合抗原。可将抗原溶解或使其呈悬浮状态,再将其加入(a)冻干状态的已成型脂质体,(b)作为溶胀溶液或悬浮液的干脂质,或(c)用于形成脂质体的脂质溶液。也可用从角质层提取的脂质(如神经酰胺和胆固醇衍生物)制成脂质体(Wertz,1992)。
氯仿是较优选的脂溶剂,但它在贮存时易变质。因而每隔1至3个月,氯仿需重蒸馏后再用作形成脂质体的溶剂。蒸馏后可加入0.7%乙醇作防腐剂。乙醇和甲醇也是适合的溶剂。
将用于形成脂质体的脂溶液置于一圆底瓶中。较优选梨形烧瓶,尤其是Lurex Scientific的产品(Vineland,新泽西,产品目录号JM-5490)。烧瓶的体积应比预期加入的脂质体水悬液体积至少大10倍,以便在形成脂质体过程中可以适当地搅动。
用旋转蒸发器,借助附于水龙头上的抽滤器,在负压下于37℃除溶剂10分钟。再将烧瓶置于干燥器中,在低真空(即低于50毫米汞柱)下进一步干燥1小时。
为将抗原包入脂质体中,可将抗原水溶液按一定体积加入至冻干的脂质体脂质中,使脂质体脂质终浓度约为200mM,并摇晃或振荡直至所有干脂质体脂质全变湿。然后可将脂质体-抗原混合液于4℃放置18至72小时。该脂质体-抗原制剂可立即使用,也可存放数年。较优选直接在经皮免疫体系中采用这种制剂,而不去除未被包入脂质体的抗原。可用水浴超声处理之类的技术使脂质体变小,以增强经皮免疫。
可不将抗原加至水溶液中,而仍按上述方法制备脂质体。再在制好的脂质体中加入抗原,因而抗原可以是溶液形式,和/或与脂质体相关,但未被其包裹。制备含有脂质体的制剂这一方法因其简便而较为优选。可用水浴超声处理之类的技术改变脂质体的大小和/或层数以增强免疫。
尽管本发明的实施并不要求,但加脂质体至制剂中可促进对角质层的水化作用。脂质体已用作载体,与佐剂共同增强对混合于、包裹于、附着于脂质体或与其关联的抗原的免疫应答。抗原的经皮送递
用本发明可以获得有效的免疫,因为抗原的经皮运送可靶向朗格汉斯细胞。这些细胞大量存在于皮肤中,是有效的抗原呈递细胞,产生T-细胞记忆,并发生有效免疫应答(Udey,1997)。由于皮肤中大量朗格汉斯细胞的存在,经皮送递的效率可能与皮肤表面接触抗原和佐剂的面积相关。事实上,经皮免疫之所以如此有效,可能就是因为它靶向的是比肌内免疫更大量的有效抗原呈递细胞。但只需少量朗格汉斯细胞或树突细胞便足以引起免疫。
我们预计,本发明将增强免疫接种方式,并诱导有效免疫应答。因为经皮免疫不涉及穿透皮肤及其复杂性和困难,对训练有素之人、无菌技术、无菌设备的需求有所降低。而且,也消除了对于在多个位点免疫或进行多次免疫的屏障。我们还预计,可通过一次施用制剂而进行免疫接种。
用单纯的抗原加佐剂溶液浸透纱布,置于封闭性补片内,或用其它补片技术,进行外表皮施用,可以完成免疫接种;还可用乳油,浸透剂,油膏和喷雾剂等其它可能的施用方法。免疫接种可由未经训练者完成,亦可自己完成。由于易于操作,故可进行大面积免疫接种。此外,一次简单的免疫接种操作即可改善对幼儿患者、成年人和第三世界国家人们的免疫接种。
类似地,动物亦可用本发明免疫。可在耳、下腹、爪、结膜、皮肤擦破处或肛门区等解剖学部位用药,或用蘸、浸等方法用药。
过去的疫苗制剂是用针注射入皮肤内的。用针注射疫苗有些缺点,如注射伴有痛感,需无菌针头和注射器,需训练有素的医务人员来完成疫苗接种,注射会引起不适,及用针穿刺皮肤会带来一些潜在的并发症。不用针而通过皮肤免疫(即经皮免疫)避免了上述缺点,因而是疫苗送递的一大进步。
本发明的经皮免疫送递体系也无需用声能或电能穿透完整皮肤。这种用电场诱导击穿角质层电介质的体系在美国专利号5,464,386中有述。
此外,经皮免疫可能优于用针免疫,因为在前一情况下,使用靶向皮肤表面大面积的多位点免疫接种,可能会靶向更多的免疫细胞。足以诱导免疫应答的治疗有效量抗原既可在单一皮肤部位、又可在覆盖多个引流淋巴结区(如颈部,腋窝,腹股沟,肱骨内上髁,腘,那些胸腹部区域)一块完整皮肤区域进行经皮送递。与在单一部位,经皮内、皮下或肌内注射少量抗原相比,在遍布全身的各部位靠近大量不同淋巴结的这种部位进行接种会对免疫体系产生更广泛刺激。
穿过或进入皮肤的抗原可能会遇到抗原呈递细胞,它们以某种方式加工抗原而诱导免疫应答。多处免疫接种可募集更大量抗原呈递细胞,而募集的更大量抗原呈递细胞群体会更强烈地诱导免疫应答。经皮免疫可在十分接近淋巴结引流处用药,因而能提高免疫接种的效率或效力。我们设想,经皮肤吸收可将抗原送递至皮肤的吞噬细胞,如皮肤树突细胞,巨噬细胞,及其他皮肤抗原呈递细胞;抗原还可被送递至肝、脾及骨髓的吞噬细胞内,已知这些细胞能通过血液或淋巴系统而充当抗原呈递细胞。结果将会是抗原被广泛分送至抗原呈递细胞,这是目前所用的免疫方法很少能达到的。
经皮免疫体系可直接施用于皮肤,并且使其风干;亦可擦入皮肤或头皮内;与敷料、补片或吸收剂一起附在皮肤上;或通过如袜子、施鞋、手套或衬衫等装置而附着;或喷在皮肤上以尽可能大面积地接触皮肤。抗原制剂可在吸收剂敷料内或纱布内施用。制剂亦可包裹在封闭性敷料内,诸如在抗原和AQUAPHOR(凡士林,矿物油,矿物蜡,羊毛蜡,泛酰醇(panthenol),bisabol和甘油,均来自Beiersdorf提供),塑料膜,浸渍聚合物,COMFEEL(Coloplast)或凡士林的乳剂中;或包裹在非封闭性敷料内,如DUODERM(3M)或OPSITE(Smith & Napheu)。封闭性敷料完全不容许水通过。部分封闭性敷料(如TEGADERM(可提供水化作用,并可使补片较长时间敷用,或防止皮肤发生浸渍。
制剂可以被施用于单一或多个位点,或者单一或多处肢体,或通过完全浸入而施用于皮肤表面大面积区域。亦可将制剂直接施用于皮肤。
基因免疫已由美国专利号5,589,466和5,593,972进行了描述。制剂中所含核酸可编码抗原、佐剂,或同时编码二者。该核酸可以能够或不能够复制;可以是非整合性的和非感染性的。该核酸中还可含有与编码抗原或佐剂之序列有效地连接在一起的调节区(如启动子、增强子、沉默子、转录启动位点和终止位点、RNA剪切受体位点和供体位点,多聚腺苷酸化信号,内部核糖体结合位点、翻译起始点和终止点)。该核酸可以与促进转染的试剂(如阳离子脂质,磷酸钙,DEAE-葡聚糖,Polybrene-DMSO,或其组合物)复合。该核酸还可含有来源于病毒基因组的区域。这些物质和技术均描述于Kriegler(1990)和Murray(1991)中。
免疫应答可包括体液效应物(即抗原特异性抗体)和/或细胞效应物(即抗原特异性淋巴细胞,如B细胞,CD4+T细胞,CD8+T细胞,CTL,Th1细胞,Th2细胞,和/或TDTH细胞)。而且免疫应答还可包括NK细胞,其介导抗体依赖性细胞介导的细胞毒性(ADCC)。
本发明制剂所诱导的免疫应答可以包括激发抗原特异性抗体和/或细胞毒性淋巴细胞(CTL,Alving和Wassef,1994中进行了综述)。可用免疫检测技术测定抗体,并可测出各种同种型(如IgM,IgD,IgA1,IgA2,分泌型IgA,IgE,IgG1,IgG2,IgG3或IgG4)。亦可用中和试验检测免疫应答。
抗体是B淋巴细胞产生的保护性蛋白质。它们具有高度特异性,通常靶向抗原的其中一个表位。在抵抗疾病的保护作用中,抗体常通过与导致该病之病原体来源的抗原发生特异性反应而起着一定的作用。免疫接种可诱导产生对该免疫抗原(如霍乱毒素)特异的抗体。当抗原经脂质体穿过皮肤送递时,可诱导产生抗原特异性抗体。
CTL是特殊的保护性免疫细胞,可抵抗病原体感染。它们也是高度专一的。免疫接种可诱导产生对抗原(如基于疟疾蛋白以及自身主要组织相容性抗原的合成多肽)特异的CTL。通过经皮免疫送递体系免疫接种诱导的CTL可杀死被病原体感染的细胞。免疫接种也可用于产生记忆性应答,表现在加强抗体和CTL的应答,抗原刺激后淋巴细胞培养物中淋巴细胞发生增殖,及皮内单用抗原刺激皮肤引起的延迟型超敏反应。
钙通道阻断剂(如利心平、Verpamil)能通过抑制抗原代谢及随后的表皮朗格汉斯细胞呈递,而抑制接触性超敏反应,估计可以利用这种钙通道阻断剂对经皮免疫诱导的T-辅助细胞应答进行操纵。经皮施用钙通道阻断剂估计会影响共同刺激分子(如B7相关家族)的表面表达及后续T辅助细胞应答的产生。估计加入钙通道阻断剂还可能会抑制延迟型超敏反应,可用于选择以细胞免疫为主的应答或是以体液免疫为主的应答。
在病毒中和试验中,将血清的连续稀释液加入宿主细胞中,然后用感染性病毒攻击,观察细胞的感染情况。或者,可将血清的连续稀释液与感染性滴度的病毒温育后再接种动物,然后观察接种动物是否出现感染症状。
本发明的经皮免疫体系可用动物或人的攻击模型进行评估,其中评估的是抗原接种保护受试者抵抗疾病的能力。有这种保护作用就证明了有抗原特异性免疫应答。攻击模型中,达到5IU/ml或更高的抗白喉抗体效价通常就认为是最佳保护作用,将此看作有保护作用的替代标志(Plotkin和Mortimer,1994)。
此外,镰状疟原虫攻击模型可用于诱导人的抗原特异性免疫应答。可对人志愿者接种含有衍生自疟疾寄生虫之寡肽或蛋白质(多肽)的经皮免疫体系,再用实验手段或经自然感染疟疾。利用Plasmodium yoelii小鼠疟疾攻击模型可以评价小鼠中抗疟病的保护作用(Wang等,1995)。
Alving等(1986)将含有脂质A的作为佐剂的脂质体注射到兔子中,以诱导对霍乱毒素(CT)及对由疟疾寡肽组成之合成蛋白质的免疫应答,所述疟疾寡肽含有与BSA偶联的4个四肽(Asn-Ala-Asn-Pro)。作者发现,与无脂质A的类似脂质体相比,将抗原包裹在含有脂质A的脂质体内,大大增强了对霍乱毒素或合成疟疾蛋白的免疫应答。现已将衍生自镰状疟原虫环子孢子蛋白(CSP)或裂殖子表面蛋白的数种抗原,包裹于含脂质A的脂质体中。包入含脂质A的脂质体中的所有疟疾抗原均显示能诱导体液免疫效应物(即抗原特异性抗体),其中一些显示还能诱导细胞介导的应答。通过免疫荧光术,对整个固定的疟疾子孢子或CTL对转染了CSP的靶细胞的杀伤力进行分析,可以测定接种了疟疾抗原的动物中产生免疫应答和免疫保护的情况。
小鼠经皮免疫霍乱毒素后可抵抗鼻内20μg霍乱毒素的攻击、Mallet等(私人通信)发现,C57B1/6小鼠鼻内用CT攻击后,产生了致死性的出血性肺炎。或者,小鼠可接受腹膜内一定剂量的CT攻击(Dragunsky等,1992)。霍乱毒素特异性IgG或IgA抗体可提供对抗霍乱毒素攻击的保护(Pierce,1978;Pierce和Reynolds,1974)。
人接受LT或CT免疫接种后,再相应用分泌LT的大肠杆菌或分泌CT的霍乱弧菌攻击,预计可产生类似的保护效果。此外,还证实在CT和LT免疫接种者及CT和LT所致疾病患者中有交叉保护现象。
如以下实施例所示,粘膜免疫力可借助于经皮接种途径获得。小鼠经皮接种CT后可检测到粘膜IgG和IgA的存在。对于病理过程发生在粘膜部位的疾疾,如LT或CT所致疾病,或病原生物体的侵染发生在粘膜部位的疾病,或粘膜感染是重要致病因素的疾病,由经皮途径引起粘膜免疫以达到保护作用可能有重要意义。
可以预计,针对流感之类疾病进行经皮免疫,由于能够诱导粘膜免疫或全身性免疫,或者体液、细胞或粘膜免疫力之组合,因而这种免疫接种可能是有效的。
疫苗可有效抵抗某些宿主效应,如通过诱导抗钳合蛋白抗体,可阻止疟疾中红细胞结合至血管内皮。
用灭活全病毒,病毒衍生的亚基或其重组产物,通过经皮途径有可能诱导产生保护性抗体,如抗甲肝、乙肝或戊肝的抗体。
经皮免疫诱导产生的抗毒素抗体可抵抗破伤风、白喉及其它毒素介导性疾病。预计可以使用含有如CT及类毒素(如破伤风和白喉等类毒素),或片段(如破伤风C片段)之类佐剂的破伤风“加强型”补片。初次免疫后,通过注射或经皮接种相同或相似抗原,可达到加强免疫。对于可诱导免疫力,但在加强免疫中有潜在负作用的注射性免疫接种,以经皮加强免疫为佳。口或鼻免疫均可用经皮途径加强。还可同时使用注射途径和经皮途径免疫。
免疫接种也已用作癌症和自身免疫疾病的治疗方法。例如,接种肿瘤抗原(如前列腺特异性抗原)后,可诱导抗体形成、CTL及淋巴细胞增殖等多种形式的免疫应答,使机体免疫体系识别并杀死肿瘤细胞。在癌症免疫治疗中,已有结果表明,靶向树突细胞(朗格汉斯细胞即为其中的特殊亚群)是一种重要战略。可用于接种的肿瘤抗原已就黑色素瘤(美国专利号5,102,663,5,141,742,和5,262,177)、前列腺癌(美国专利号5,538,866)和淋巴瘤(美国专利号4,816,249,5,068,177和5,277,159)进行了描述。接种T细胞受体寡肽可诱导产生免疫应答,使自身免疫疾病的进程终止(美国专利号5,612,035和5,614,192;Antel等,1996;Vandenbark等,1996)。美国专利号5,552,300也描述了适于治疗自身免疫疾病的抗原。
下文意在举例说明本发明,但本发明的实施不受这些实施例的任何限制。
实施例免疫接种操作
用40#剪刀对6-8周龄BALB/C小鼠剃毛。剃毛过程应不对皮肤造成任何损伤。剃毛范围从胸廓中部至紧贴颈背下部。让小鼠休息24小时。此前已对小鼠作了耳部标记以便辩认,并已预先取血以获得免疫前血清。也在未去毛小鼠的每只耳朵处施用多达50μl免疫溶液,对其进行经皮免疫。
然后按如下方法免疫小鼠。用20mg/ml甲苯噻嗪溶液0.03-0.06ml,100mg/ml氯胺酮0.5ml麻醉小鼠;这一剂量的麻醉剂使小鼠静止不动约1小时。将小鼠腹面朝下置于温暖的毯子上。
将免疫溶液按以下方法加到小鼠的去毛背部皮肤上:将1.2cm×1.6cm的镂空聚苯乙烯板轻轻放在小鼠背部,用浸透盐水的无菌纱布部分湿润皮肤(可用免疫溶液),然后用滴管将免疫溶液加在镂空板处,形成2cm2的免疫溶液补片。或者,在剃毛部位或耳部均匀地涂上固定体积的免疫溶液。小心使滴管尖不刮伤或擦伤皮肤。用滴管尖较光滑的一侧将免疫溶液在欲覆盖的区域内涂开。
使免疫溶液(约100μl至200μl)在小鼠背部静置60至180分钟。60分钟后,轻轻抓住小鼠的脖子和尾部,用大量微温的自来水冲洗10秒钟。然后用一块无菌纱布将小鼠轻轻拍干,再洗10秒;再次拍干后将小鼠放回笼中。小鼠看起来对麻醉、免疫接种、洗涤过程或外毒素的毒性没表现出任何不适反应。免疫接种后,未见皮肤刺激或红肿,小鼠生存状态很好。用耳免疫方法同上,只是不必在免疫接种前去毛。抗原
用以下抗原进行免疫接种和ELISA测定,并将抗原与无菌PBS或生理盐水混合。霍乱毒素或CT(List Biologicals,目录号101B,批号10149CB),CT的B亚基(List Biologicals,目录号BT01,批号CVXG-14E),CT的A亚基(List Biologicals,目录号102A,批号CVXA-17B),CT的A亚基(Calbiochem,目录号608562);百日咳毒素(不含盐)(List Biologicals,批号181120a);破伤风类毒素(List Biologicals,批号1913a和1915a);假单胞菌外毒素A(List Biologicals,批号ETA25a);白喉类毒素(List Biologicals,批号15151);大肠杆菌不耐热肠毒素(Sigma,批号9640625);牛血清白蛋白或BSA(Sigma,目录号3A-4503,批号31F-0116);流感嗜血杆菌B偶联物(Connaught,批号6J81401)。ELISA-IgG(H+L)
在类似于Glenn等(1995)的技术中,利用ELISA方法,测定CT、LT、ETA、百日咳毒素、白喉类毒素、破伤风类毒素、流感嗜血杆菌B偶联物、流感病毒、钳合蛋白和BSA的特异性抗体。所有抗原均溶于无菌盐溶液中,浓度为2μg/ml。按每孔50μl(0.1μg),将溶液加到IMMULON-2聚苯乙烯平板(Dynatech Laboratories,Chantilly,VA)上,并在室温温育过液。然后用0.5%酪蛋白/0.05%吐温20封闭缓冲溶液封闭平板1小时。血清用0.5%酪蛋白/0.05%吐温20稀释剂稀释;在平板上的各栏中对各稀释度进行处理。在室温温育2小时。
然后用PBS-0.05%吐温20洗涤溶液洗涤平板4次,按1/500的稀释度,将山羊抗小鼠IgG(H+L)辣根过氧化物酶(HRP)连接的(Bio-Rad Laboratories,Richmond,CA,目录号170-6516)二抗稀释于酪蛋白稀释剂中,加在平板上,于室温1小时。然后在PBS-吐温洗涤溶液中洗涤平板4次。每孔中加入100μl 2,2’-连氮-双(3-乙基苯并噻唑啉)磺酸底物(Kirkegaard和Perry),显色20-40分钟后在405nm的波长下读取平板的吸光度。结果表示为各份血清的几何平均值及ELISA单位(吸光度相当于1.0的血清稀释度)平均值的标准误差或ELISA单位形式的各种抗体应答。ELISA-IgG(γ),IgM(μ)和IgA(α)
在类似于Glenn等(1995)的技术中,利用ELISA方法,测定IgG(γ),IgM(μ)和IgA(α)抗CT抗体。将CT溶于无菌盐溶液中,浓度为2μg/ml。按每孔50μl(0.1μg),将溶液加到IMMULON-2聚苯乙烯平板(Dynatech Laboratories,Chantilly,VA)上,并在室温温育过液。然后用0.5%酪蛋白/0.05%吐温20封闭缓冲溶液封闭平板1小时。血清用酪蛋白稀释剂稀释;在平板上进行连续稀释。在室温温育2小时。
然后用PBS-吐温洗涤溶液洗涤平板4次,按1/1000的稀释度,将山羊抗小鼠IgG(γ)辣根过氧化物酶(HRP)连接的(Bio-RadLaboratories,Richmond,CA,目录号172-1038)、山羊抗小鼠IgM(μ)HRP连接的(BioRad Laboratories,Richmond,CA,目录号172-1030)或山羊抗小鼠IgA HRP连接的(Sigma,St.Louis,MO,目录号1158985)二抗稀释于酪蛋白稀释剂中,加在平板上,于室温1小时。然后在PBS-吐温洗涤溶液中洗涤平板4次。每孔中加入100μl 2,2’-连氮-双(3-乙基苯并噻唑啉)磺酸底物(Kirkegaard和Perry,Gaithersburg,MD),在405nm的波长下读取平板的吸光度。结果表示为各份血清的几何平均值及ELISA单位(吸光度相当于1.0的血清稀释度)平均值的标准误差。ELISA-IgG亚类
如Glenn等(1995)所述,测定抗CT、LT、ETA和BSA的抗原特异性IgG亚类(IgG1,IgG2a,IgG2b和IgG3)。在IMMULON-2聚苯乙烯板(Dynatech Laboratories,Chantilly,VA)上进行固相ELISA。每孔分别加入相应抗原的盐溶液(0.1μg/50μl),温育过夜,并用0.5%酪蛋白-吐温20封闭。对稀释于0.5%酪蛋白中的每份小鼠血清进行连续稀释,再在室温下温育4小时。二抗由辣根过氧化物酶偶联的山羊抗小鼠同种型特异性抗体(IgG1,IgG2a,IgG2b,IgG3,The Binding Site,San Diego,CA)组成。用小鼠骨髓瘤IgG1、IgG2a、IgG2b和IgG3(The Binding Site,San Diego,CA),测定每亚类的标准曲线。标准孔用山羊抗小鼠IgG(H+L)(Bio-Rad Laboratories,Richmond,CA,目录号172-1054)包被,以捕获以连续稀释度加入的骨髓瘤IgG亚类标准品。也用过氧化物酶偶联的山羊抗小鼠亚类特异性抗体检测骨髓瘤IgG亚类。待检血清和骨髓瘤标准品均用2,2’-连氮-双(3-乙基苯并噻唑啉)磺酸(Kinkegaard和Preey,Gaithersburg,MD)作底物进行检测。读取405nm的吸收值。用对骨髓瘤标准品曲线计算出的线性滴定曲线上的值定量测定每种抗原特异性亚类,以μg/ml的形式表示。ELISA-IgE
抗原特异性IgE抗体的定量测定按Pharmingen TechnicalProtocls,Research Products目录,第541页,1996-1997(Pharmingen,San Diego,CA)的程序进行。将于0.1M NaHCO3(pH8.2)中的2μg/ml纯化抗小鼠IgE捕获单克隆抗体(mAb)(Pharmingen,目录号02111D)50μl加入IMMUNO平板(Nunc,目录号12-565-136)上。将平板在室温温育过夜,用PBS-吐温20洗3次,用含3%BSA的PBS溶液封闭2小时,再用PBS-吐温洗3次。血清用含1%BSA的PBS稀释,以1/100的梯度加入,并在各栏中连续稀释(如1/100,1/200等等)。加入纯化的小鼠IgE标准品(Pharmingen,目录号0312D),起始稀释度为0.25μg/ml,并在各栏中连续稀释。将平板温育2小时,再用PBS-吐温洗涤5次。
将生物素标记的抗小鼠IgE单克隆抗体(Pharmingen,目录号02122D)溶于含1%BSA的PBS中,浓度为2μg/ml,温育45分钟,再用PBS-吐温洗涤5次。加入抗生物素蛋白标记的过氧化物酶(Sigma A3151,1mg/ml溶液的1/400稀释液)30分钟后,用PBS-吐温洗涤平板6次。待检血清和IgE标准品均用2,2’-连氮-双(3-乙基苯并噻唑啉)磺酸(Kirkegaard和Perry,Gaithersburg,MD)作底物进行检测。读取405nm处的吸收值。用对IgE标准品曲线计算出的线性滴定曲线上的值定量测定各种抗原特异性亚类,表示为μg/ml。脂质体制备
若经皮免疫用制剂中含有脂质体,则根据Alving等(1993)的方法制备由二肉豆蔻酰基磷脂酰胆碱、二肉豆蔻酰基磷脂酰甘油、胆固醇组成的多层脂质体。这三种试剂购自Avanti Polar Lipids Inc.(Alabaster,AL)。脂质的氯仿贮存液贮于-20℃冰箱中,使用时从中取出。
按0.9∶0.1∶0.75的摩尔比将二肉豆蔻酰基磷脂酰胆碱,二肉豆蔻酰基磷脂酰甘油和胆固醇在一梨形瓶内混合。用旋转蒸发器,于37℃,在负压条件下处理10分钟去除溶剂。再将烧瓶置于干燥器内,低真空下进一步干燥2小时以去除残余溶剂。用无菌水将脂质体溶胀成37mM磷脂,再冻干并存于-20℃。将这些脂质体以其冻干状态与生理盐水(pH7.0)混合,以获得盐溶液中的预计磷脂浓度。或者,也可将干燥脂质溶胀于生理盐水(pH7.0)中,以形成脂质体,且不再冻干。
实施例1
按上文“免疫接种方法”中所述对5只小鼠一组的6至8周龄BALB/c小鼠进行经皮免疫。使用100μl按下述制备的免疫接种溶液免疫小鼠:将按上文“脂质体制备”中所述制备的脂质体与盐溶液混合以形成脂质体,然后用盐溶液(仅含脂质体的组)或溶于盐溶液中的CT稀释预先形成的脂质体,以产生免疫溶液,每100μl所述溶液中含有含10-150mM磷脂的脂质体和100μg CT。将CT与盐溶液混合,以制备每100μl溶液含100μg CT的免疫溶液,供仅接受CT的组使用。接种前将溶液旋涡处理10秒钟。
在第0和3周对小鼠进行经皮免疫,加强免疫3周后,使用上文“ELISA IgG(H+L)”中所述的ELISA测定抗体水平,并与免疫前的血清相比较。如表1所示,除使用150mM脂质体的小鼠外,由无脂质体的CT诱导的抗CT抗体水平与使用脂质体产生的抗CT抗体水平没有区别。仅盐溶液中的CT就能免疫小鼠以抗CT,从而产生高的抗体滴度。
表1.抗CT抗体
组 | ELISA单位 | SEM |
仅施用CT | 27,482 | (16,635-48,051) |
CT+150mM脂质体 | 4,064 | *(2,845-5,072) |
CT+100mM脂质体 | 35,055 | (25,932-44,269) |
CT+50mM脂质体 | 9,168 | (4,283-12,395) |
CT+25mM脂质体 | 18,855 | (12,294-40,374) |
CT+10mM脂质体 | 28,660 | (18,208-31,498) |
50mM脂质体 | 0 |
*与仅施用CT的组显著不同(P<0.05)
实施例2
按上文“免疫接种方法”中所述对5只小鼠一组的6至8周龄BALB/c小鼠进行经皮免疫。在第0和3周使用100μl按下述制备的免疫接种溶液免疫小鼠:将BSA与盐溶液混合以制备每100μl盐溶液含200μg BSA的免疫溶液,供仅接受BSA的组使用;在盐溶液中将BSA和CT混合以制备每100μl盐溶液含200μg BSA和100μg CT的免疫溶液,供接受BSA和CT的组使用。当使用脂质体时,按上文“脂质体制备”中所述制备脂质体,并首先与盐溶液混合以形成脂质体,然后用于盐溶液中的BSA或BSA+CT稀释所形成的脂质体以产生免疫溶液,每100μl免疫溶液中含有含50mM磷脂的脂质体,还含有200μg BSA,或每100μl免疫溶液中还含有200μg BSA+100μgCT。接种前将溶液旋涡处理10秒钟。
第二次免疫3周后,使用上文“ELISA IgG(H+L)”中所述的ELISA测定血清中的抗体,结果示于表2。仅用BSA,含或不含脂质体都不能引发抗体应答,然而,加入CT即可刺激对BSA的免疫应答。CT作为对BSA之免疫应答的佐剂起作用,产生了高滴度的抗BSA抗体。
表2.抗BSA抗体
组 | ELISA单位 | SEM |
盐溶液中的BSA | 0 | |
BSA+50mM脂质体 | 0 | |
盐溶液中的CT+BSA | 8,198 | (5,533-11,932) |
CT+BSA+50mM | 3,244 | (128-3,242) |
实施例3
按上文“免疫接种方法”中所述对5只小鼠一组的6至8周龄BALB/c小鼠进行经皮免疫。在第0和3周使用100μl按下述制备的免疫溶液免疫小鼠:将LT与盐溶液混合以制备每100μl盐溶液含100μg LT的免疫溶液供仅接受LT的组使用。当使用脂质体时,按上文“脂质体制备”中所述制备脂质体,并首先与盐溶液混合以形成脂质体,然后用盐溶液中的LT稀释预先形成的脂质体以产生免疫溶液,每100μl免疫溶液中含有含50mM磷脂的脂质体和100μg LT。接种前将溶液旋涡处理10秒钟。
第二次免疫3周后,使用上文“ELISA IgG(H+L)”中所述的ELISA测定抗LT抗体,结果示于表3。含或不含脂质体的LT显然都有免疫原性,在不同的组之间未检测到显著差异。LT和CT是细菌ADP-核糖基化外毒素(bARE)家族的成员,它们以A∶B酶原的形式组合在一起,其中A亚基中含有ADP-核糖基转移酶活性,B亚基的功能是与靶细胞结合。在氨基酸水平上,LT与CT 80%同源,两者具有相似的非共价结合的亚基组合、化学计量(A∶B5),相同的结合靶点,神经节苷脂GM1,和相似的大小(MW约为80,000)。对CT和LT具有相似量的抗体应答(表1和3)反映出LT和CT的相似性似乎在经皮途径中影响到它们的免疫原性。
表3.抗LT抗体
组 | ELISA单位 | SEM |
盐溶液中的LT | 23,461 | (20,262-27,167) |
LT+50mM脂质体 | 27,247 | (19,430-38,211) |
实施例4
按上文“免疫接种方法”中所述对5只小鼠一组的6至8周龄C57B1/6小鼠进行经皮免疫。使用100μl按下述制备的免疫溶液免疫小鼠1次:将LT与盐溶液混合以制备每100μl盐溶液含100μg LT的免疫溶液。接种前将溶液旋涡处理10秒钟。
单次免疫3周后,使用上文“ELISA IgG(H+L)”中所述的ELISA测定抗LT抗体,结果示于表4。单次免疫中,LT明显为免疫原性的,抗体的产生持续3周。抗体滴度的快速增加和对单次免疫接种的反应可能是经皮免疫方法的有用方面。可以想象得到:快速的单次免疫对于流行病、旅行者和就医较难的地方是有用的。
表4.抗LT抗体
小鼠号 | ELISA单位 |
5141 | 6,582 |
5142 | 198 |
5143 | 229 |
5144 | 6,115 |
5145 | 17,542 |
几何平均值 | 2,000 |
实施例5
按上文“免疫接种方法”中所述对5只小鼠一组的8至12周龄C57B1/6小鼠进行经皮免疫。使用100μl按下述制备的免疫溶液免疫接种小鼠1次:将CT与盐溶液混合以制备每100μl盐溶液含100μgCT的免疫溶液。接种前将溶液旋涡处理10秒钟。
单次免疫3周后,使用上文“ELISA IgG(H+L)”中所述的ELISA测定抗CT抗体,结果示于表5。单次免疫中,CT即具有高免疫原性。抗体滴度的快速增加和对单次免疫接种的应答可能是经皮免疫方法的有用方面。可以想象得到:快速的单次免疫对于流行病、旅行者和就医较难的地方是有用的。
表5.抗CT抗体
小鼠号 | ELISA单位 |
2932 | 18,310 |
2933 | 30,878 |
2934 | 48,691 |
2935 | 7,824 |
几何平均数 | 21,543 |
实施例6
按上文“免疫接种方法”中所述对5只小鼠一组的6至8周龄BALB/c小鼠进行经皮免疫。在第0和3周使用100μl按下述制备的免疫溶液免疫小鼠:将ETA与盐溶液混合以制备每100μl盐溶液含100μg ETA的免疫溶液供仅接受ETA的组使用。当使用脂质体时,按上文“脂质体制备”中所述制备脂质体,并首先与盐溶液混合以形成脂质体。然后用盐溶液中的ETA稀释预先形成的脂质体以产生免疫溶液,每100μl免疫溶液中含有含50mM磷脂的脂质体和100μgETA。接种前将溶液旋涡处理10秒钟。
第二次免疫3周后,使用上文“ELISA IgG(H+L)”中所述的ELISA测定血清中的抗体,结果示于表6。含或不含脂质体的ETA显然都有免疫原性,在不同的组之间未检测到显著差异。ETA与CT和LT的不同之处在于ETA是613个氨基酸的单肽,A和B结构域位于同一肽上,且ETA与完全不同的受体,即α2-巨球蛋白受体/低密度脂蛋白受体相关蛋白(Kounnas等,1992)结合。尽管ETA与CT在大小、结构和结合靶点上没有相似性,但ETA也可诱导经皮的抗体应答。
表6.抗-ETA抗体
组 | ELISA单位 | SEM |
盐溶液中的ETA | 3,756 | (1,926-7,326) |
ETA+50mM脂质体 | 857 | (588-1,251) |
实施例7
按上文“免疫接种方法”中所述对5只小鼠一组的6至8周龄BALB/c小鼠进行经皮免疫。使用100μl按下述制备的免疫溶液免疫小鼠:将CT与盐溶液混合以制备每100μl盐溶液含100μg CT的免疫溶液,将LT与盐溶液混合以制备每100μl盐溶液含100μg LT的免疫溶液,将ETA与盐溶液混合以制备每100μl盐溶液含100μg ETA的免疫溶液,将CT和BSA一起与盐溶液混合以制备每100μl盐溶液含100μg CT和200μg BSA的免疫溶液。接种前将溶液旋涡处理10秒钟。
在第0和3周对小鼠进行经皮免疫,加强免疫3周后,使用上文“ELISA IgG亚类”中所述的ELISA测定抗体水平,并与免疫前的血清相比较。对CT、BSA和LT的IgG亚类应答产生了相似的IgG1和IgG2a水平,这反映出激活了Th1和Th2淋巴细胞中的T辅助细胞(Seder和Paul,1994),而对ETA的IgG亚类应答几乎全部由IgG1和IgG3组成,这与Th2样应答一致(表7)。由此看来使用经皮免疫可产生所有的IgG亚类。
表7.诱导抗体的IgG亚类
免疫抗原 | 抗体特异性 | IgG1(μg/μl) | IgG2a(μg/μl) | IgG2b(μg/μl) | IgG3(μg/μl) |
CT | CT | 134 | 25 | 27 | 0 |
CT+BSA | BSA | 108 | 17 | 12 | 5 |
LT | LT | 155 | 28 | 10 | 8 |
ETA | ETA | 50 | 0 | 1 | 10 |
实施例8
按上文“免疫接种方法”中所述对5只小鼠一组的6至8周龄BALB/c小鼠进行经皮免疫。使用100μl按下述制备的免疫溶液免疫小鼠:将LT与盐溶液混合,以制备每100μl盐溶液含100μg LT的免疫溶液,供仅接受LT的组使用;将CT与盐溶液混合,以制备每100μl盐溶液含100μg CT的免疫溶液,供仅接受CT的组使用;将ETA与盐溶液混合,以制备每100μl盐溶液含100μg ETA的免疫溶液,供仅接受ETA的组使用;将BSA和CT与盐溶液混合,以制备每100μl盐溶液含100μg BSA和100μg CT的免疫溶液,供接受BSA和CT的组使用。
在第0和3周对小鼠进行经皮免疫,加强免疫1周后,使用上文“ELISA IgE”中所述的ELISA测定抗体水平,并与免疫前血清相比较。如表8所示,尽管检测的灵敏度为0.003μg/ml,但仍未发现IgE抗体。第二次免疫3周后,使用“ELISA IgG(H+L)”测定相同小鼠血清中的IgG抗体。对LT、ETA、CT和BSA的IgG抗体应答示于下文,结果表明动物成功地被免疫,并以高滴度的抗体对各个抗原作出应答。
表8.对LT,ETA,CT和BSA的IgE抗体
组 | 抗体特异性 | IgE(μg/ml) | IgG(ELISA单位) |
LT | 抗LT | 0 | 23,461 |
ETA | 抗ETA | 0 | 3,756 |
CT | 抗CT | 0 | 39,828 |
CT+BSA | 抗BSA | 0 | 8,198 |
实施例9
按上文“免疫接种方法”中所述对5只小鼠一组的6至8周龄BALB/c小鼠进行经皮免疫。在第0和3周使用100ml按下述制备的免疫溶液免疫小鼠:将CT与盐溶液混合以制备每100ml体积含100mg CT的免疫溶液。接种前将免疫溶液旋涡处理10秒钟。
在第0和3周对小鼠进行经皮免疫,并使用上文“ELISA IgG(H+L)”和“ELISA IgG(γ)”中所述的ELISA测定抗体水平。初次免疫1和4周后进行检测,并与免疫前血清比较。如表9所示,盐溶液中的CT诱导了高水平的抗CTIgG(γ)抗体。使用IgM(μ)特异性第二抗体可检测到少量的IgM。在4周内,抗体应答主要是IgG。数据以ELISA单位表示。
表9.IgG(γ)和IgM(μ)
免疫组 | 周 | IgG(γ) | IgM(μ) |
CT | 1 | 72 | 168 |
CT | 4 | 21,336 | 38 |
L()+CT | 1 | 33 | 38 |
L()+CT | 4 | 22,239 | 70 |
实施例10
按上文“免疫接种方法”中所述对5只小鼠一组的6至8周龄BALB/c小鼠进行经皮免疫。使用100μl按下述制备的免疫溶液免疫小鼠1次:将CT与盐溶液混合以制备每100μl盐溶液含100μg CT的免疫溶液。接种前将溶液旋涡处理10秒钟。在第0和3周对小鼠进行经皮免疫。加强免疫5周后,使用上文“ELISA IgG(H+L)”中所述的ELISA测定抗体水平,并与免疫前血清比较。如表10所示,检测到血清抗CT的IgA抗体。
表10.抗CT的IgA抗体
小鼠号 | IgA(ng/ml) |
1501 | 232 |
1502 | 22 |
1503 | 41 |
1504 | 16 |
1505 | 17 |
实施例11
按上文“免疫接种方法”中所述对5只小鼠一组的6至8周龄BALB/c小鼠进行经皮免疫。使用100μl按下述制备的免疫溶液免疫小鼠:将CT与盐溶液混合以制备每100μl体积含100μg CT的免疫溶液。接种前将免疫溶液旋涡处理10秒钟。
在第0和3周用100μl免疫溶液对小鼠进行经皮免疫,并使用上文“ELISA IgG(H+L)”和“ELISA IgG(γ)”中所述的ELISA测定抗体水平。初次免疫8周后进行抗体检测并与免疫前血清比较。如表11所示,盐溶液中的CT诱导了高水平的血清抗CT抗体。使用IgG(H+L)或IgG(γ)特异性抗体,通过ELISA可检测到肺洗液中的IgG。由于通过用于收集粘膜抗体的灌洗法稀释了在肺粘膜表面发现的抗体,因此,检测到的抗体确切量不如可测抗体的存在本身那么重要。
处死小鼠后得到肺洗液,小心解剖以暴露气管和肺,在气管分叉上方横切气管。插入22号聚丙烯管,结扎气管以在边缘形成密闭的封口。使用与管贴附的1ml注射器灌注0.5ml PBS,液体使肺缓缓充胀。取出液体重新灌注,共进行3次灌洗,然后将肺洗液置于-20℃冻存。
表11表示在第8周,血清和肺洗液中针对霍乱毒素的IgG(H+L)和IgG(γ)抗体应答。数据以ELISA单位表示。所有小鼠的肺洗液中都可清楚地检测到抗体。粘膜中抗体的存在对于针对粘膜活性疾病的保护作用可能是很重要的。
表11.针对CT的粘膜抗体
动物号 免疫组 IgG(H+L) IgG(γ) 来源
1501 CT 133 34 肺
1502 CT 75 12 肺
1503 CT 162 28 肺
1504 CT 144 18 肺
1505 CT 392 56 肺
几何平均值 156 26
1501 CT 34,131 13,760 血清
1502 CT 11,131 2,928 血清
1503 CT 21,898 10,301 血清
1504 CT 22,025 8,876 血清
1505 CT 34,284 10,966 血清
几何平均值 23,128 8,270
实施例12
在第0和3周,按上文“免疫接种方法”中所述对4只小鼠一组的BALB/c小鼠进行经皮免疫。按上文“脂质体制备”中所述制备脂质体,并首先与盐溶液混合以形成脂质体,然后用盐溶液中的CT、CTA或CTB稀释预先形成的脂质体以产生免疫溶液,每100μl免疫溶液中含有含50mM磷脂的脂质体和50μg抗原(CT、CTA或CTB)。接种前将溶液旋涡处理10秒钟。
加强免疫1周后,使用上文“ELISA IgG(H+L)”中所述的ELISA测定抗体,并与免疫前血清相比较,结果示于表12。CT和CTB显然是免疫原性的,而CTA则无免疫原性,因此,CT的B亚基是必需的,并足以诱导强的抗体应答。
表12.针对CT、CTA和CTB的抗体
组 | 抗CT抗体 | 抗CTA抗体 | 抗CTB抗体 |
CT+50mM脂质体 | 12,636 | 136 | 7,480 |
CTB+50mM脂质体 | 757 | 20 | 1,986 |
CTA+50mM脂质体 | 0 | 0 | 0 |
实施例13
按上文“免疫接种方法”中所述对5只小鼠一组的BALB/c小鼠进行经皮免疫。在第0和3周,用每100μl盐溶液中含有100μg白喉类毒素和10μg百日咳毒素的免疫溶液免疫小鼠。接种前将溶液旋涡处理10秒钟。
使用上文“ELISA IgG(H+L)”中所述的ELISA定量测定抗体。仅在用百日咳毒素和白喉类毒素都免疫过的动物中检测到抗白喉类毒素的抗体。应答最大的小鼠中抗白喉类毒素抗体的ELISA单位为1038。因此,少量百日咳毒素是作为白喉类毒素抗原的佐剂起作用的。仅有类毒素不能诱导免疫应答,这暗示着类毒素处理已影响到分子中负责ADP-核糖基化外毒素中所发现的佐剂效应的部分。
表13.针对白喉的抗体
小鼠号 免疫抗原 IgG ELISA单位
4731 DT+PT 1,039
4732 DT+PT 1
4733 DT+PT 28
4734 DT+PT 15
4735 DT+PT 20
4621 DT 0
4622 DT 0
4623 DT 0
4624 DT 0
4625 DT 0
实施例14
按上文“接种方法”中所述对5只小鼠一组的BALB/c小鼠进行经皮免疫。在第0、8和20周,用每100μl盐水中含有50μg百日咳毒素(List,目录号181,批号181-20a)的免疫溶液免疫小鼠1次。
使用上文“ELISA IgG(H+L)”中所述的ELISA定量测定抗体。在最后一次加强免疫用百日咳毒素免疫过的动物1周后,检测到抗百日咳毒素的抗体。最后一次免疫后,所有5只动物的抗百日咳毒素抗体的水平都有所提高。因此,百日咳毒素可作为其自身的佐剂起作用,并可诱导PT特异性的IgG抗体。在如白喉/百日咳/破伤风/Hib的联合疫苗中,PT的佐剂效应对于增强针对共同施用的抗原以及针对PT自身的抗体应答都是有用的。
表14.针对百日咳毒素的抗体应答
小鼠号 抗原 2周 21周
5156 PT 14 256
5157 PT 22 330
5158 PT 17 303
5159 PT 33 237
5160 PT 75 418
实施例15
按上文“免疫接种方法”中所述对5只小鼠一组的BALB/c小鼠进行经皮免疫。在第0周,用每100μl盐溶液中含有50μg破伤风类毒素和100μg霍乱毒素的免疫溶液免疫小鼠1次。
使用上文“ELISA IgG(H+L)”中所述的ELISA定量测定抗体。第8周时,在动物5173中检测到的抗破伤风类毒素抗体为443ELISA单位。
实施例16
使用125I-标记的CT示踪抗原/佐剂的去处,以评价上表皮施药和随后清洗施药位点之后的饲养过程中发生口服免疫的可能性。麻醉小鼠,按上文“免疫接种方法”中所述,用100μg经125I-标记的CT(150,000cpm/μg CT)对小鼠进行经皮免疫。对照小鼠被持续麻醉6小时以免除饲养,将实验小鼠麻醉1小时,清洗后进行饲养,至第6小时时,处死小鼠,将器官称重并在Packard gamma计数器上计数125I。在免疫位点处的剃毛皮肤上检测到总量为2-3μg的CT(14,600cpm/μg组织),而在胃(661cpm/μg组织)和肠(9cpm/μg组织)中最多检测到0.5μg的CT。
在第0和3周,用10μg于盐溶液中的CT(不含NaHCO3)进行口服免疫(n=5),诱导出的6周平均IgG抗体应答<1,000ELISA单位,而如上所述,用100μg CT进行经皮免疫可导致清洗后的皮肤中残留低于5μg的CT,第6周时的抗CT应答为42,178ELISA单位。诱导对口服CT的免疫应答需要在免疫溶液中加入NaHCO3(Piece,1978;Lycke和Holmgren,1986),因此,当将CT经表皮施用于皮肤时,口服免疫对检测到的抗体没有显著的贡献。
实施例17
使用经上表皮施用于皮肤、具体为小鼠耳(其中易于观察到大群体的朗格汉斯细胞(Enk等,1993;Bacci等,1997))的于盐溶液中的霍乱毒素(CT),并对在激活的朗格汉斯细胞中被上调的主要组织相容性复合体(MHC)II类分子进行染色(Shimada等,1987),得到了朗格汉斯细胞激活的体内证据。
当小鼠被麻醉时,用100μg于盐溶液中的CT,100μg于盐溶液中的CTB或仅用盐溶液覆盖BALB/c小鼠耳的背侧1小时,或对阳性对照皮内注射100pg LPS或10μg TNFα。然后彻底清洗耳,24小时后,摘下耳,如Caughman等人(1986)所述收集表皮块,并对MHC II类表达进行染色。用MKD6(抗I-Ad)或阴性对照Y3P(抗I-Ak)对表皮块进行染色,并将山羊抗小鼠FITC F(ab)2用作第二步试剂。先前已发现,耳上经皮免疫的小鼠(如上文所述,未剃毛)在单次免疫3周后具有7,000ELISA单位的抗CT抗体。
相对于对照而言,在用CT和CTB免疫过的小鼠的表皮块中,通过染色强度检测到MHC II类分子表达增强,朗格汉斯细胞数目降低(尤其是用霍乱毒素时),并且朗格汉斯细胞形态学有变化(图1),这表明经表皮施用霍乱毒素激活了朗格汉斯细胞(Aiba和Katz,1990;Enk等,1993)。
实施例18
朗格汉斯细胞代表了被称为“树突细胞”的有效佐细胞家族的表皮群体。人们认为朗格汉斯细胞(可能还有真皮中的相关细胞)是针对皮肤所遭遇的外源抗原的免疫应答所必需的。朗格汉斯细胞之“生命周期”的特征在于至少有两个完全不同的时期。表皮中的朗格汉斯细胞(“岗哨”)可摄取颗粒,并有效地处理抗原,但却是未接触抗原的T细胞的弱刺激物。与之形成对照的是,在与表皮抗原接触之后被诱导迁移至淋巴结的朗格汉斯细胞(“信使”)吞噬能力差,处理抗原的能力有限,但却是原初T细胞的强有力刺激物。如果朗格汉斯细胞既要充当其“岗哨”的角色,又要充当其“信使”的角色,它们必需能在表皮中存留,暴露于抗原之后也能以受控的方式离开表皮,因此,朗格汉斯细胞-角质形成细胞粘附作用的调节代表朗格汉斯细胞运输和功能中的一个关键控制点。
朗格汉斯细胞表达E-钙粘着蛋白(B1auvelt等,1995),该蛋白是突出体现于上皮的同嗜性粘着分子。角质形成细胞也表达此粘着分子,在体外,E-钙粘着蛋白明显介导鼠朗格汉斯细胞对角质形成细胞的粘着。已知E-钙粘着蛋白参与朗格汉斯细胞在表皮中的定位,见Stingl等人(1989)有关朗格汉斯细胞和角质形成细胞之鉴定和特性的综述。
已知表皮朗格汉斯细胞(LC)的迁移及其将抗原从皮肤转运至引流淋巴结在皮肤免疫应答(如接触致敏)的诱导中是至关重要的。在至淋巴结的转运过程中,朗格汉斯细胞经受了多种表型变化,这些变化是它们从皮肤处移动和获得抗原呈递能力所必需的。除了MHCII类分子的上调外,调节与周围组织基质和T淋巴细胞之间相互作用的粘着分子的表达也有变化。已知朗格汉斯细胞的迁移与E-钙粘着蛋白表达的显著降低(Schwarzenberger和Udey,1996)和ICAM-1的平行上调(Udey,1997)相关。
细菌ADP核糖基化外毒素(bARE)的经皮免疫靶向表皮中的朗格汉斯细胞。bARE激活朗格汉斯细胞,将其从岗哨的角色转化为信使的角色。摄入的抗原随即被转运至淋巴结并呈递给B和T细胞(Streilein和Grammer,1989;Kripke等,1990;Tew等,1997)。在此过程中,表皮的朗格汉斯细胞成熟为淋巴结中呈递抗原的树突细胞(Schuler和Steinman,1985);进入淋巴结的淋巴细胞分离为B细胞滤泡和T细胞区域。已知激活朗格汉斯细胞以使其成为可迁移的朗格汉斯细胞不仅与MHC II类分子的显著增加有关,还与E-钙粘着蛋白表达的显著降低和ICAM-1的上调有关。
我们预计,霍乱毒素(CT)及其B亚基(CTB)可上调ICAM-1的表达并下调朗格汉斯细胞上E-钙粘着蛋白的表达,以及上调朗格汉斯细胞上MHC II类分子的表达。CT或CTB通过使岗哨朗格汉斯细胞自由移动以呈递如BSA或白喉类毒素之类的抗原而行使佐剂的作用,所述抗原在与用作佐剂的CT或CTB相遇的相同地点和时间被朗格汉斯细胞吞噬。从表皮细胞或朗格汉斯细胞自身释放的包括TNFα和IL-1β的细胞因子可介导朗格汉斯细胞的激活,以上调ICAM-1的表达并下调E-钙粘着蛋白的表达。
预计这种经皮免疫辅助法对可激活朗格汉斯细胞的任何化合物都起作用。激活可以下调E-钙粘着蛋白和上调ICAM-1的方式发生。随后,朗格汉斯细胞可将由这种朗格汉斯细胞激活化合物和抗原(如白喉类毒素或BSA)的混合物制成的抗原携至淋巴结,在其中,抗原被呈递给T细胞并引发免疫应答。因此,如bARE之类的激活物质通过激活朗格汉斯细胞以吞噬如白喉类毒素的抗原,迁移至淋巴结,成熟为树突细胞并将抗原呈递给T细胞,即可用作其它经皮非免疫原性的抗原(如白喉类毒素)的佐剂。
细胞因子和/或趋化因子的使用可影响对经皮免疫中所用抗原的T辅助细胞应答。例如,白细胞介素-10(IL-10)可将抗体应答歪曲为Th2 IgG1/IgE应答,而抗IL-10可增强IgG2a的产生(Bellinghausen等,1996)。
实施例19
钳合蛋白是在受疟疾感染的红细胞表面表达的分子,其作用是将疟疾寄生的红血细胞锚定于血管内皮。这是寄生虫存活所必需的,并对死于脑型疟疾之儿童中镰状疟原虫疟疾的病理发生有直接作用。在脑型疟疾中,由于钳合蛋白分子与宿主内皮细胞受体CD36的特异性相互作用,脑毛细管被大量寄生的红血细胞堵塞。Ockenhouse等人鉴定出了宿主受体CD36和介导此受体-配体相互作用的寄生分子(钳合蛋白)。Ockenhouse等人已将与CD36受体相互作用的钳合蛋白分子的结构域克隆和表达成由大肠杆菌产生的重组蛋白。在下文实施例中使用截短的79个氨基酸的钳合蛋白产物。
用重组钳合蛋白或编码钳合蛋白之基因的DNA进行活性免疫应该能引发抗体,所述抗体能阻断被疟疾寄生的红细胞与宿主内皮细胞CD36的粘着,从而阻止寄生虫生命周期的完成,因其不能与内皮细胞结合而导致寄生虫死亡。策略是开发一种可引发高滴度阻断抗体的免疫方法,这种方法之一是经皮送递疫苗。对总抗体滴度以及阻断活性和调理作用的测量构成这种经皮免疫方法的基础。本实验中所用的重组钳合蛋白长度为79个氨基酸(约18KDa),并含有分子的CD36结合结构域。我们还构建了裸露DNA构建体,该构建体由此结合域组成,用表皮基因枪送递法引发了抗体。
按上文“免疫接种方法”中所述对BALB/c小鼠(n=3)进行经皮免疫。在第0和8周使用120μl按下述制备的免疫溶液免疫小鼠:将编码镰状疟原虫钳合蛋白的质粒与盐溶液混合,以制备每100μl盐溶液含80μg质粒、80μg CT(List Biologicals)的免疫溶液。用醇棉签(Triad Alcohol pad,70%异丙醇)小心地清洗耳之后,将120μl免疫溶液施用于未经标记的耳上。不通过清洗除去免疫溶液。
在初次免疫3,4,7和9周后,从尾静脉收集血清,使用上文“ELISA IgG(H+L)”中所述的ELISA测定血清中的钳合蛋白抗体,结果示于表15。
第二次加强免疫后,钳合蛋白DNA与CT一起诱导了可测的针对表达蛋白的抗体应答。为了引起免疫,该蛋白质需被免疫系统表达和加工,因此,对于针对由编码钳合蛋白之质粒表达的钳合蛋白的免疫应答而言,CT行使的是佐剂的作用。
在非人灵长类动物中,已显示出DNA疫苗可引发针对如疟疾(Gramzinski,疫苗,15:913-915,1997)和HIV(Shriver等,疫苗,15:884-887,1997)之类疾病的中和性抗体,并在几个实验模型中显示出不同程度的保护作用(McClements等,疫苗,15:857-60,1997)。预期通过皮肤进行DNA免疫所引发的应答类似于靶向皮肤免疫系统的基因枪所引发的应答(Prayaga等,疫苗,15:1349-1352,1997)。
表15.经钳合蛋白(Seq)DNA和霍乱毒素(CT)
免疫过的动物中针对Seq蛋白的血清抗体
动物号 | 免疫组 | IgG(H+L)ELISA单位 | |||
第3周 | 第4周 | 第7周 | 第9周 | ||
8966 | Seq DNA/CT | 58 | 80 | 33 | - |
8967 | Seq DNA/CT | 76 | 81 | 41 | 146 |
8968 | Seq DNA/CT | 54 | 33 | 26 | - |
几何平均数 | 62 | 60 | 33 | ||
预先取血的收集物 | - | 40 |
实施例20
按上文“免疫接种方法”中所述,使用钳合蛋白对5只小鼠一组的BALB/c小鼠进行经皮免疫。在第0、2和8周,使用100μl按下述制备的免疫溶液免疫小鼠:在第0周,用410μl中含有59μg CT和192μg钳合蛋白的免疫溶液对接受钳合蛋白和CT之组的小鼠进行免疫,用410μl中含有192μg钳合蛋白的免疫溶液对仅接受钳合蛋白之组的小鼠进行免疫,用520μl中含有120μg CTB和250μg钳合蛋白的免疫溶液对接受钳合蛋白和CTB之组的小鼠进行免疫。2周后,用含有163μg钳合蛋白的345μl盐溶液对仅接受钳合蛋白之组的小鼠进行加强免疫,用含有163μg钳合蛋白和60μg CT的345μl盐溶液对接受CT和钳合蛋白之组的小鼠进行加强免疫,用含有163μg钳合蛋白和120μg CTB的345μl盐溶液对接受钳合蛋白和CTB之组的小鼠进行加强免疫。在第二次加强免疫中,对仅接受钳合蛋白之组的小鼠施用120μg钳合蛋白,对接受CT和钳合蛋白之组的小鼠施用120μg钳合蛋白和120μg CT,对接受钳合蛋白和CTB之组的小鼠施用120μg钳合蛋白和120μg CTB。
在初次免疫3、5、7、9、10、11和15周后,使用上文“ELISAIgG(H+L)”中所述的ELISA测定抗体,结果示于表16。仅用钳合蛋白只诱导少量的但可测的抗体应答。然而,加入CT可刺激出强得多的针对钳合蛋白的免疫应答,加入CTB可诱导出强于仅用钳合蛋白所诱导的免疫应答。CT和CTB作为针对身为重组蛋白之钳合蛋白的免疫应答的佐剂起作用。
表16 Seq,Seq+霍乱毒素(CT),Seq+霍乱毒素B(CTB)
IgG(H+L)ELISA单位动物号 免疫组 检测抗原 预先取血 第3周 第5周 第7周 第8周 第9周 第11周 第15周2861 Seq Seq 7 7 20 32 709 431 4082862 Seq Seq 8 5 14 136 33 4 62863 Seq Seq 8 63 38 393 467 348 4592864 Seq Seq 5 9 26 102 32 13 112865 Seq Seq 9 19 76 111 100 53 98
几何平均值 9 13 29 114 129 54 652866 Seq/CT Seq 923 1145 125 639 43679 28963 429812867 Seq/CT Seq 73 84 154 ND 9428 20653 274032868 Seq/CT Seq 805 370 1447 1105 ND 13169 76772869 Seq/CT Seq 175 760 1317 768 113792 118989 2700402870 Seq/CT Seq 153 158 535 241 3245 ND 4277
几何平均值 271 336 456 601 19747 31115 252792871 Seq/CTB Seq 8 3 87 40 22 29 1922872 Seq/CTB Seq 4 6 24 22 35 24 342873 Seq/CTB Seq 107 138 128 51 2074 2283 22962874 Seq/CTB Seq 6 7 22 18 41 40 4572875 Seq/CTB Seq 515 504 1910 1744 ND 7148 5563
几何平均值 25 25 102 68 91 214 520汇集物 5
实施例21
按上文“免疫接种方法”中所述,对5只小鼠一组的BALB/c小鼠进行经皮免疫。在第0周,使用100μl按下述制备的免疫溶液免疫小鼠:将FLUSHIELD(Wyeth-Ayerst,纯化的亚病毒颗粒,1997-98制剂,批号U0980-35-1)冻干,并与盐溶液混合,以制备每100μl盐溶液含有90μg FLUSHIELD亚病毒颗粒的免疫溶液,供仅接受流感病毒的组使用;在盐溶液中将流感病毒和CT混合,以制备每100μl盐溶液含有90μg FLUSHIELD抗原和100μg CT的免疫溶液,供接受流感病毒和CT的组使用。
在初次免疫3周后,使用上文“ELISA IgG(H+L)”中所述的ELISA测定抗体,结果示于表17。仅用流感病毒不能诱导抗体应答。然而,加入CT可刺激出比仅用流感病毒所观察到的强得多的免疫应答,因此,在针对FLUSHIELD、亚病毒颗粒流感疫苗、得自病毒之抗原混合物的免疫应答中,CT是作为佐剂起作用的。
表17.在仅用流感病毒(Inf)或用Inf+霍乱毒素(CT)
免疫过的动物中针对甲型和乙型流感病毒的血清抗体
动物号 | 免疫组 | IgG(H+L) |
ELISA单位 | ||
第3周 | ||
8601 | CT/Inf | 144 |
8602 | CT/Inf | 14 |
8603 | CT/Inf | 1325 |
8604 | CT/Inf | 36 |
8605 | CT/Inf | 29 |
几何平均值 | 77 | |
8606 | Inf | 17 |
8607 | Inf | 16 |
8608 | Inf | 20 |
8609 | Inf | 23 |
8610 | Inf | 23 |
几何平均值 | 20 |
实施例22
LT是ADP-核糖基化外毒素家族的成员,其分子量类似于CT,LT与神经节苷脂GM1结合,与CT具有80%的同源性,并具有相似的A∶B5化学计量。因此,在经皮免疫中,LT也被用作DT的佐剂。在第0,8和18周,按上述用每100μl盐溶液含有100μg LT(Sigma,目录号E-8015,批号17hH12000)和100μg CT(List Biologicals,目录号101b)的盐溶液免疫BALB/c小鼠(n=5)。如表18所示,LT诱导了中等程度的抗DT应答。
ETA(List Biologicals,批号ETA 25A)是ADP-核糖基化外毒素家族的成员,但它是与不同受体结合的单个多肽。按上文所述,在第0、8和18周,在100μl含有100μg CT的盐溶液中将100μg ETA送递至BALB/c小鼠的背部。在第20周,ETA加强了抗DT应答,因此,其它ADP-核糖基化外毒素也能作为共同施用的蛋白质的佐剂起作用(表18)。表18.在用铜绿假单胞菌外毒素A(ETA)和DT或大肠杆菌不耐热肠毒素(LT)和DT免疫过的动物中白喉类毒素(DT)抗体滴度的动力学
动物号 | 免疫组 | 检测抗原 | IgG(H+L) | ELISA单位 |
预先取血 | 第20周 | |||
5146 | ETA/DT | DT | 31718 | |
5147 | ETA/DT | DT | 48815 | |
5148 | ETA/DT | DT | 135 | |
5149 | ETA/DT | DT | 34 | |
5150 | ETA/DT | DT | 258 | |
几何平均数 | 1129 | |||
5136 | LT/DT | DT | 519 | |
5137 | LT/DT | DT | 539 | |
5138 | LT/DT | DT | 38 | |
5139 | LT/DT | DT | 531 | |
5140 | LT/DT | DT | 901 | |
几何平均数 | 348 | |||
汇集物 | 3 |
实施例23
按上文“免疫接种方法”中所述对5只小鼠一组的BALB/c小鼠进行经皮免疫。在第0、8和18周,用100μl含有100μg霍乱毒素(List Biologicals,目录号101B,批号10149CB),50μg破伤风类毒素(List Biologicals,目录号191B,批号1913a和1915b)和83μg白喉类毒素(List Biologicals,目录号151,批号15151)的盐溶液免疫小鼠。
使用上文“ELISA IgG(H+L)”中所述的ELISA定量测定针对CT、DT和TT的抗体。在初次免疫后23周检测抗CT、DT或TT的抗体。所有经免疫小鼠中抗白喉类毒素和霍乱毒素的抗体水平均有升高。反应最大的小鼠中抗破伤风类毒素抗体的ELISA单位为342,约为在未免疫动物之血清中所检测抗体水平的80倍。因此,不相关抗原的联合(CT/TT/DT)可用于针对各个抗原的免疫。这表明霍乱毒素可用作多价疫苗的佐剂。
表19.用霍乱毒素、破伤风类毒素和白喉类毒素
同时免疫的动物中的血清抗体
动物号 | 免疫组 | 检测抗原 | IgG(H+L) | ELISA单位 |
预先取血 | 第23周 | |||
5176 | CT/TT/DT | CT | 7636 | |
5177 | CT/TT/DT | CT | 73105 | |
5179 | CT/TT/DT | CT | 126259 | |
5216 | CT/TT/DT | CT | 562251 | |
5219 | CT/TT/DT | CT | 66266 | |
汇集物 | ≤3 | |||
几何平均值 | 76535 | |||
5176 | CT/TT/DT | DT | 64707 | |
5177 | CT/TT/DT | DT | 17941 | |
5179 | CT/TT/DT | DT | 114503 | |
5216 | CT/TT/DT | DT | 290964 | |
5219 | CT/TT/DT | DT | 125412 | |
汇集物 | ≤4 | |||
几何平均值 | 86528 | |||
5176 | CT/TT/DT | TT | 21 | |
5177 | CT/TT/DT | TT | 30 | |
5179 | CT/TT/DT | TT | 342 | |
5216 | CT/TT/DT | TT | 36 | |
5219 | CT/TT/DT | TT | 30 | |
汇集物 | ≤2 | |||
几何平均值 | 47 |
实施例25
使用CT经皮免疫可诱导强有力的免疫应答,使用CT作为佐剂和抗原,将肌内注射和口服免疫的免疫应答与经皮免疫的相比较。使用200μl取液器吸头将溶于25μl盐溶液中的25μg CT(ListBiologicals,目录号101b)经口服施用于BALB/c小鼠(n=5)。小鼠易于吞咽免疫溶液。按上文所述对经皮标记组中小鼠的耳施用盐溶液中的1mg/ml CT 25μl。给肌内标记组小鼠的前腿肌内注射25μg于盐溶液中的CT。
肌内注射了CT的小鼠在注射部位显示出明显的肿胀和触痛,并产生了高水平的抗CT抗体。经皮免疫的小鼠在免疫部位未出现红或肿胀,并产生了高水平的抗CT抗体。相对于经皮免疫的小鼠而言,经口服免疫的小鼠所产生抗体的水平低得多。这表明,在经皮免疫的小鼠中,通过饲喂进行口服免疫不是经皮免疫所诱导的高水平抗体的原因。总的说来,经皮免疫途径优于口服或肌内免疫,因为经皮免疫可得到高水平的抗体,而且不会对免疫产生不良反应。
表20.通过经皮、口服或肌内途径免疫的动物中
霍乱毒素抗体滴度的动力学
动物号 | 免疫途径 | IgG(H+L) | ELISA单位 |
预先取血 | 第6周 | ||
8962 | 经皮免疫 | 23489 | |
8963 | 经皮免疫 | 30132 | |
8964 | 经皮免疫 | 6918 | |
8965 | 经皮免疫 | 20070 | |
8825 | 经皮免疫 | 492045 | |
汇集物 | 16 | ||
几何平均值 | 34426 | ||
8951 | 口服 | 743 | |
8952 | 口服 | 4549 | |
8953 | 口服 | 11329 | |
8954 | 口服 | 1672 | |
汇集物 | 14 | ||
几何平均值 | 2829 | ||
8955 | 肌内免疫 | 35261 | |
8958 | 肌内免疫 | 607061 | |
8959 | 肌内免疫 | 452966 | |
8850 | 肌内免疫 | 468838 | |
8777 | 肌内免疫 | 171648 | |
汇集物 | 12 | ||
几何平均值 | 239029 |
实施例26
按上文“免疫接种方法”中所述,对5只小鼠一组的BALB/c小鼠进行经皮免疫。在第0、8和20周,使用100μl按下述制备的免疫溶液免疫小鼠:将Hib偶联物(Connaught,批号6J81401,86μg/ml)冻干以浓缩抗原,将冻干的产物与盐溶液混合,以制备每100μl盐溶液含有50μg Hib偶联物的免疫溶液,供仅接受Hib偶联物的组使用;在盐溶液中将Hib偶联物和CT混合,以制备每100μl盐溶液含有50μg Hib偶联物和100μg CT的免疫溶液,供接受Hib偶联物和CT的组使用。
在第二次免疫3周后,使用上文“ELISA IgG(H+L)”中所述的ELISA测定血清抗体,结果示于表21。仅用Hib偶联物能诱导少量但可测的抗体应答。然而,加入CT刺激出了强得多的针对Hib偶联物的免疫应答。CT在针对Hib偶联物的免疫应答中是作为佐剂起作用的。这表明,通过本文所述的方法,可将多糖偶联物抗原用作经皮抗原。
表21.针对流感嗜血杆菌b(Hib)的抗体
动物号 | 免疫组 | IgG(H+L)ELISA单位 |
5211 | Hib | 57 |
5212 | Hib | 29 |
5213 | Hib | 28 |
5214 | Hib | 63 |
5215 | Hib | 31 |
几何平均值 | 39 | |
5201 | CT/Hib | 1962 |
5202 | CT/Hib | 3065 |
5203 | CT/Hib | 250 |
5204 | CT/Hib | 12 |
5205 | CT/Hib | 610 |
几何平均值 | 406 | |
预先取血的汇集物 | - | 1 |
实施例27
乳剂、乳膏和凝胶对于免疫化合物在皮肤表面、头发或身体皱褶处方便地涂抹具有实际可行的优点。另外,这种制剂可能还有能增强免疫效力的优点,如封闭或水合作用。
使用得自大肠杆菌的不耐热肠毒素(LT)(Sigma,目录号E-8015,批号17hH1200)来比较使用单纯盐溶液及公知的石油基软膏-AQUAPHOR进行经皮免疫的效力,所述软膏“可以单独使用,或实质上使用水溶液与任何软膏混合使用,或与其它油基物质和所有公知的局部药物联合使用”(p507 PDR,非处方药物,1994,第15版)。用一系列剂量处理小鼠,以评价对比较性赋形剂中降低剂量的相对抗体应答。
除在背部施用免疫溶液3小时外,按上述免疫BALB/c小鼠。分别使用2mg/ml,1mg/ml,0.5mg/ml或0.2mg/ml的溶液制备LT盐溶液,以送递50μl剂量的溶液和溶于溶液中的100μg、50μg、25μg或10μg抗原。3小时后,使用湿纱布轻擦背部以除去免疫溶液。
按下述制备油包水制剂:在具有luer锁紧套口的1ml结核菌素玻璃注射器中,使用与两个注射器相连的15号乳化针头将等体积的AQUAPHOR和盐溶液中的抗原混合直至混合物混合均匀。分别使用盐溶液中的4mg/ml、2mg/ml、1mg/ml或0.5mg/ml的LT溶液与等体积的AQUAPHOR混合。将50μl此混合物施用于被剃光的背部3小时,然后用纱布擦拭以小心除去所述混合物。称量含有LT的油包水乳剂中的抗原剂量以给药50μl。通过将盐溶液的比重(1.00g/ml)和AQUAPHOR的比重0.867gm/ml相加,总和除以2得到终比重0.9335gm/ml,可计算出重量/体积比。约47mg含有LT的油包水乳剂被施用于小鼠以进行免疫。
对盐溶液和油包水乳剂送递的LT均有明显的剂量-反应关系(表22),100μg诱导了最高水平的抗体,10μg诱导了较低但有效的免疫应答。油包水乳化的LT诱导的应答类似于盐溶液中的LT,似乎为经皮免疫提供了便利的送递机制。类似地,也可使用凝胶、乳膏或更加复杂的制剂,如油包水包油制剂,为经皮免疫送递抗原。这种组合物也可与补片、封闭性补片或储器一起使用,并且可以长期或短期使用免疫抗原和佐剂。表22.经盐溶液中的LT或AQUAPHOR乳剂中的LT不同剂量免疫的动
物中针对大肠杆菌不耐热肠毒素(LT)的血清抗体
免疫 | IgG(H+L)Elisa单位 | IgG(H+L)Elisa单位 | ||||||
组 | 乳剂 | 动物号 | 预先取血 | 第3周 | 乳剂 | 动物号 | 预先取血 | 第3周 |
LT 100μg | 盐溶液 | 8741 | 18434 | Aquaphor | 8717 | 6487 | ||
LT 100μg | 盐溶液 | 8742 | 16320 | Aquaphor | 8719 | 4698 | ||
LT 100μg | 盐溶液 | 8743 | 19580 | Aquaphor | 8774 | 18843 | ||
LT 100μg | 盐溶液 | 8744 | 19313 | Aquaphor | 8775 | 18217 | ||
LT 100μg | 盐溶液 | 8745 | 22875 | Aquaphor | 8861 | 16230 | ||
汇集物 | 32 | 汇集物 | 54 | |||||
几何平均值 | 19190 | 11117 | ||||||
LT 50μg | 盐溶液 | 8736 | 19129 | Aquaphor | 8721 | 4160 | ||
LT 50μg | 盐溶液 | 8737 | 3975 | Aquaphor | 8722 | 12256 | ||
LT 50μg | 盐溶液 | 8738 | 6502 | Aquaphor | 8725 | 12262 | ||
LT 50μg | 盐溶液 | 8739 | 6224 | Aquaphor | 8771 | 12982 | ||
LT 50μg | 盐溶液 | 8740 | 18449 | Aquaphor | 8772 | 15246 | ||
汇集物 | 54 | 汇集物 | 57 | |||||
几何平均值 | 8929 | 10435 | ||||||
LT 25μg | 盐溶液 | 8768 | 3274 | Aquaphor | 8727 | 3585 | ||
LT 25μg | 盐溶液 | 8731 | 3622 | Aquaphor | 8728 | 3 | ||
LT 25μg | 盐溶液 | 8732 | 557 | Aquaphor | 8729 | 4206 | ||
LT 25μg | 盐溶液 | 8733 | 626 | Aquaphor | 8862 | 7353 | ||
LT 25μg | 盐溶液 | 8734 | 1725 | Aquaphor | 8769 | 5148 | ||
汇集物 | 56 | 汇集物 | 53 | |||||
几何平均值 | 1481 | 1114 | ||||||
LT 10μg | 盐溶液 | 8848 | 621 | Aquaphor | 8748 | 1968 | ||
LT 10μg | 盐溶液 | 8849 | 475 | Aquaphor | 8749 | 1935 | ||
LT 10μg | 盐溶液 | 8757 | 858 | Aquaphor | 8750 | 646 | ||
LT 10μg | 盐溶液 | 8759 | 552 | Aquaphor | 8747 | 1569 | ||
LT 10μg | 盐溶液 | 8760 | 489 | Aquaphor | 8764 | 1 | ||
汇集物 | 43 | 汇集物 | 39 | |||||
几何平均值 | 585 | 329 |
实施例28
按上文“免疫接种方法”中所述对5只小鼠一组的小鼠进行经皮免疫。在第0、8和18周,用100μl仅含有50μg破伤风类毒素(List Biologicals,目录号191B,批号1913a和1915b)和83μg白喉类毒素(List Biologicals,目录号151,批号15151),或还含有100μg霍乱毒素(List Biologicals,目录号101B,批号10149CB)的盐溶液免疫小鼠。
使用上文“ELISA IgG(H+L)”中所述的ELISA定量测定抗白喉类毒素的抗体。在用TT/DT或CT/TT/DT免疫过的动物中检测到抗类毒素抗体的水平有所提高。然而,在使用CT作为佐剂的动物中,抗体滴度高很多。在每个再次免疫(8和18周)之后,两组的抗类毒素抗体滴度都明显增加。因此,尽管DT可诱导针对其自身的少量但明显的应答,但是,1)霍乱毒素作为佐剂加入和2)用佐剂霍乱毒素和抗原(白喉类毒素)加强免疫可增加应答的量。典型的加强免疫应答依赖于T细胞记忆,在此实验中,抗DT抗体的加强表明通过经皮免疫使T-细胞参与到应答中。
表23.经破伤风类毒素(TT)和白喉类毒素(DT)或霍乱毒素(CT)、TT
和DT免疫的动物中DT抗体滴度的动力学
IgG(H+L)ELISA单位动物号 免疫组 检测抗原 预先取血 第2周 第4周 第6周 第8周 第10周 第14周 第17周 第18周 第20周 第23周5196 TT/DT DT 7 12 18 23 49 56 33 18 219 1665197 TT/DT DT 5 11 11 10 15 17 16 17 125 755198 TT/DT DT 13 20 16 - 28 25 27 7 48 1725199 TT/DT DT 13 8 10 10 11 22 12 217 178 2635200 TT/DT DT 4 10 4 7 120 149 127 - 17309 14537
几何平均值 7 12 10 11 31 38 29 26 332 3825176 CT/TT/DT DT 8 26 21 14 3416 5892 1930 1826 63087 647045177 CT/TT/DT DT 8 6 7 8 424 189 149 175 16416 179415179 CT/TT/DT DT 8 3 4 8 4349 1984 2236 1921 124239 1145035216 CT/TT/DT DT 12 5 9 11 3238 2896 2596 1514 278281 2909645219 CT/TT/DT DT 8 15 13 12 5626 4355 2036 1941 343161 125412
几何平均值 9 8 9 10 2582 1945 1277 1125 104205 86528汇集物 4
实施例29
按上文所述,用CT(不含叠氮化物,Calbiochem)在C57B1/6小鼠被剃光的背部对小鼠进行经皮免疫。免疫后6周,使用致死攻击模型攻击小鼠(Mallet等,在小鼠霍乱毒素鼻内攻击模型中霍乱弧菌毒素(CTK63)和大肠杆菌不耐热毒素(LTK63)之无毒性突变体的免疫预防效力,提交给Immunology Letters)。在攻击中,用20μl氯胺酮-甲苯噻嗪麻醉小鼠,在麻醉状态下用塑料取液器吸头将溶于盐溶液的20μg CT(Calbiochem,不含叠氮化物)鼻内施用于小鼠。在试验#1中,14天后,15只经免疫小鼠中的12只在攻击中存活,9只未经免疫的对照小鼠中只有1只存活。因麻醉使5只对照小鼠在攻击前丧失。攻击#1中的小鼠具有15,000 ELISA单位(几何平均数)的抗CT血清抗体,在攻击时被处死的5只经免疫小鼠的肺洗液中都可检测到IgG。按上文所述收集肺洗液。
对C57B1/6幼鼠重复免疫和攻击,16只经免疫小鼠中的7只在攻击中存活,而17只未经免疫的小鼠中只有2只在攻击中存活。攻击#2中的经免疫小鼠具有41,947 ELISA单位(几何平均数)的抗CTIgG抗体。在攻击时被处死的5只小鼠的肺洗液中可检测到抗CT IgG和IgA(表24)。9只小鼠中的8只在其粪便样品中可检测到抗CT IgG和IgA(表25)。收集在攻击时自发排便之动物的新鲜粪便样品,将粪便置于-20℃冷冻。进行ELISA时,将粪便解冻,用100μl PBS匀浆,离心,对上清液进行ELISA。经免疫小鼠总的存活率是19/31或61%,而未经免疫之小鼠的总存活率为3/26或12%。
表24.肺洗液中抗霍乱毒素IgG和IgA抗体滴度样品稀释度 动物号
8969 8970 8971 8972 8995
IgG(H+L)抗CT (光密度)1∶10 3.613 3.368 3.477 3.443 3.3501∶20 3.302 3.132 3.190 3.164 3.1661∶40 3.090 2.772 2.825 2.899 2.6921∶80 2.786 2.287 2.303 2.264 2.0861∶160 2.041 1.570 1.613 1.624 1.4411∶320 1.325 0.971 1.037 1.041 0.9651∶640 0.703 0.638 0.601 0.644 0.5831∶1280 0.434 0.382 0.350 0.365 0.364
IgA抗CT (光密度)1∶2 1.235 2.071 2.005 2.115 1.9841∶4 1.994 1.791 1.836 1.85 1.8011∶8 1.919 1.681 2.349 1.796 1.7421∶16 1.8 1.457 1.577 1.614 1.5361∶32 1.503 1.217 1.36 1.523 1.231∶64 1.189 0.863 1.044 1.101 0.881∶128 0.814 0.57 0.726 0.74 0.5951∶356 0.48 0.334 0.436 0.501 0.365
表25.粪便抗霍乱毒素IgG和IgA抗体滴度
小鼠号(免疫组)样品稀释度 8985 8997 8987 8990 8977 8976 8975 8988 8994 8979 9000 8983
(CT) (CT) (CT) (CT) (CT) (CT) (CT) (CT) (无) (无) (无) (无)
IgG(H+L) 抗CT (光密度)1∶10 1.01 1.91 2.33 0.03 0.74 1.98 1.20 1.45 0.09 0.05 0.02 0.181∶20 0.42 0.94 1.26 - 0.31 1.19 0.50 0.91 0.04 - - 0.081∶40 0.20 0.46 0.68 - 0.12 0.58 0.24 0.49 - - - 0.021∶80 0.10 0.21 0.34 - 0.05 0.31 0.09 0.25 - - - -1∶160 0.03 0.09 0.18 - 0.02 0.14 0.05 0.12 - - - -
IgA抗CT(光密度)1∶4 0.32 1.14 0.43 0.00 0.19 1.00 0.58 1.21 0.02 - 0.07 -1∶8 0.16 0.67 0.24 - 0.08 0.56 0.36 0.77 - - - -1∶16 0.08 0.33 0.11 - 0.03 0.27 0.17 0.40 - - - -1∶32 0.06 0.16 0.05 - 0.03 0.12 0.08 0.20 - - - -1∶64 0.01 0.07 0.03 - - 0.05 0.03 0.10 - - - -
实施例30
C57B1/6雌性小鼠得自Charles River实验室。用200μg卵白蛋白(OVA)(Sigma,批号#14H7035,在PBS中的储存浓度为2mg/ml)和50μg霍乱毒素(List Biologicals,批号#101481B,储存浓度为5mg/ml)免疫小鼠。使用Packard Cobra Gamma计数器(#102389系列)测定所释放的51Cr的量。
用0.03ml氯胺酮-甲苯噻嗪麻醉C57B1/6小鼠,在不损伤皮肤的情况下用推剪剃光背部,让小鼠休息24小时。将小鼠麻醉,然后在第0和28天将150μl免疫溶液置于2cm2面积的剃光皮肤上2小时以免疫小鼠,再用湿纱布将小鼠擦2次。麻醉、免疫或清洗步骤未对小鼠产生不良影响。每周重复这些步骤,共重复3周。
加强免疫后1周收集脾淋巴细胞,在RPMI-1640和10%FBS(含青霉素-链霉素,谷氨酰胺,非必需氨基酸,丙酮酸钠和2-巯基乙醇)中将细胞体外培养6天,其间加入5%的大鼠伴刀豆球蛋白A上清液作为IL-2的来源,加入或不加入抗原。靶细胞仅由同系(H-2b)EL4细胞组成,将EL4细胞与CTL肽SINFEKKL,同种异型(H-2k)L929细胞和EG7细胞一起脉冲。每孔用0.1mCi 51Cr(Na2CrO4,Amersham)将靶细胞(1×106个细胞/孔)标记1小时,按3∶1至100∶1的比例加入效应细胞。于37℃,5% CO2的潮湿气氛中,在96孔圆底组织培养板(Costar,目录号#3524)中,用0.2ml完全RPMI-1640,10%FBS培养基将细胞混合物培养5小时。5小时培养结束时,用棉纱布条吸收上清液,对其进行处理以测定51Cr的释放。按下述测定比裂解:
%比裂解=100×[(实验释放值-自发释放值)/(最大释放值-自发释放值)]。
如表26所示,在经CT+OVA免疫的组中,E∶T比例为100∶1的经EL4肽脉冲的细胞中可检测到第1部分CTL。如果比裂解百分比不超过10%或不明显超过培养基刺激的效应细胞裂解背景百分比,则CTL检测为非阳性。类似地,如表26所示,在经CT+OVA免疫的组中,E∶T比例为100∶1的EG7细胞(被OVA转染的细胞)中可检测到第2部分CTL。因此,CT由经皮途径辅助产生CTL。
表26.经皮诱导的OVA特异性CTL
第1部分-靶细胞:EL4+肽
免疫组 | ||||||
CT+OVA | CT+OVA | CT | CT | OVA | OVA | |
刺激源 | ||||||
E∶T比例 | 培养基 | Ova | 培养基 | Ova | 培养基 | Ova |
100∶1 | 9.5 | 13.1 | 11.1 | 12.5 | 23.1 | 21.5 |
30∶1 | 6.9 | 6.8 | 5.9 | 8.9 | 14.2 | 10.7 |
10∶1 | 4.9 | 3.5 | 3.5 | 8.5 | 7.7 | 5.2 |
第2部分-靶细胞:EG7(转染了OVA)
免疫组 | ||||||
CT+OVA | CT+OVA | CT | CT | OVA | OVA | |
刺激源 | ||||||
E∶T比例 | 培养基 | Ova | 培养基 | Ova | 培养基 | Ova |
100∶1 | 10.6 | 17.6 | 14.5 | 16.8 | 23.8 | 26 |
30∶1 | 4.9 | 9.5 | 8.2 | 10.1 | 13.6 | 10.7 |
10∶1 | 6.4 | 4.4 | 4 | 5 | 7.3 | 4.2 |
实施例31
按上文“免疫接种方法”中所述对C57B1/6小鼠(n=6)进行经皮免疫。在第0和4周,用100μl含有100μg霍乱毒素(ListBiologicals,目录号#101B,批号# 10149CB)和250μg卵白蛋白(Sigma,鸡蛋白蛋白,Grade V目录号#A5503,批号#14H7035)的盐溶液免疫小鼠。
在第一次免疫后8周,由收集自动物的脾脏制备单细胞悬浮液。以8×105个细胞/孔的浓度,将脾细胞置于200μl含有所示浓度的OVA蛋白或不相关的伴白蛋白的培养基中培养。于37℃,在CO2培养箱中将培养物培养72小时,培养的同时向每孔中加入0.5μCi/孔的3H胸苷。12小时后,收获培养板,通过液体闪烁计数定量测定掺入的放射性标记的胸苷以评估增殖情况。3H掺入量的粗略值以cpm表示,各个样品的左边示出了倍数增长(实验cpm/培养基中的cpm)。倍数增长大于3被认为是显著的。
仅当脾细胞被卵白蛋白刺激时才可检测到显著增殖,事实上诸动物原先已被卵白蛋白体内免疫,而未经不相关的伴白蛋白免疫。因此,用霍乱毒素和卵白蛋白经皮免疫在体外诱导了脾细胞发生抗原特异性增殖,这表明此方法引发了细胞免疫应答。
表27.经霍乱毒素(CT)和卵白蛋白(OVA)
免疫之动物的脾细胞的抗原特异性增殖
免疫 | 体外刺激物浓度 | 培养基 | OVA蛋白 | ||
伴白蛋白组 | 3H掺入量cpm | 增长倍数 | 3H掺入量cpm | 增长倍数 | |
CT/OVA | 10μg/ml1427 | 13450 | 9.4 | 3353 | 2.3 |
1μg/ml | 4161 | 2.9 | 2638 | 1.8 | |
0.1μg/ml | 2198 | 1.5 | 2394 | 1.7 | |
0.01μg/ml | 3419 | 2.4 | 2572 | 1.8 |
本说明书中提及的所有专利以及其它所有公开文献的全部内容都作为参考文献全文引入本文。这些参考文献是作为本领域技术人员的指示被提及的。
尽管就目前认为可行和优选的实施方案描述了本发明,但应懂得本发明并不局限于或受限于已公开的实施方案,相反,本发明欲在所附权利要求书的主旨和范围之内覆盖多种修饰和等价替代。
因此,应懂得所述发明的变化对于本领域技术人员而言是显而易见的,它不背离本发明新的方面,这种变化欲包括在下文权利要求书的范围之内。
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Claims (36)
1.含有抗原和佐剂的经皮免疫制剂,其中给完整的皮肤施用所述制剂即可诱导特异于所述抗原的免疫应答,而无需穿透皮肤。
2.权利要求1的制剂,其中还包括敷料以形成经皮免疫补片。
3.权利要求2的制剂,其中敷料是封闭性敷料。
4.权利要求2的制剂,其中敷料覆盖了1个以上引流淋巴结区域。
5.权利要求1的制剂,其中佐剂增强了对淋巴细胞的抗原呈递。
6.权利要求1的制剂,其中佐剂激活了抗原呈递细胞。
7.权利要求6的制剂,其中抗原呈递细胞是朗格汉斯细胞或皮肤树突细胞。
8.权利要求1的制剂,其中佐剂增强了抗原呈递细胞上主要组织相容性复合物II类分子的表达。
9.权利要求8的制剂,其中抗原呈递细胞是朗格汉斯细胞或皮肤树突细胞。
10.权利要求1的制剂,其中佐剂导致施用位点下的抗原呈递细胞迁移至引流淋巴结。
11.权利要求10的制剂,其中抗原呈递细胞是朗格汉斯细胞或皮肤树突细胞。
12.权利要求1的制剂,其中佐剂给朗格汉斯细胞送递信号使之成熟为树突细胞。
13.权利要求1的制剂,其基本上由抗原和佐剂组成。
14.权利要求1的制剂,其中制剂中一种组分既是抗原又是佐剂。
15.权利要求1的制剂,所述制剂是水溶液。
16.权利要求1的制剂,所述制剂中不包含有机溶剂。
17.权利要求1的制剂,所述制剂中不包含穿透增强剂。
18.权利要求1的制剂,其被制成乳剂的形式。
19.权利要求1的制剂,其中抗原来源于选自由细菌、病毒、真菌和寄生虫组成之组的病原体。
20.权利要求1的制剂,其中抗原是肿瘤抗原。
21.权利要求1的制剂,其中抗原是自身抗原。
22.权利要求1的抗原,其中抗原是变应原。
23.权利要求1的制剂,其中抗原的分子量大于500道尔顿。
24.权利要求1的制剂,所述制剂包括至少两种不同的分离的抗原。
25.权利要求1的制剂,其中以编码抗原之核酸的形式提供抗原。
26.权利要求25的制剂,其中所述核酸是非整合的和非复制的。
27.权利要求25的制剂,其中所述核酸另外还包括与编码抗原之序列有效相连的调节区域。
28.权利要求25的制剂,所述制剂不包含穿透增强剂、病毒颗粒、脂质体或带电脂质。
29.权利要求1至28中任一项的制剂,其中佐剂是ADP-核糖基化外毒素。
30.权利要求29的制剂,其中ADP-核糖基化外毒素是霍乱毒素或其功能性衍生物。
31.权利要求29的制剂,其中ADP-核糖基化外毒素是大肠杆菌不耐热肠毒素、百日咳毒素或其功能性衍生物。
32.权利要求29的制剂,其中以编码ADP-核糖基化外毒素的核酸形式提供佐剂。
33.权利要求1至28中任一项的制剂,其中制剂的施用不涉及损伤完整皮肤的物理能、电能或声能。
34.权利要求1至28中任一项的制剂,其中免疫应答不是变应性反应、皮炎或特应性反应。
35.前述权利要求中任一项所述制剂的制备方法。
36.前述权利要求中任一项所述制剂在非人动物中诱导免疫应答的用途。
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US08/896,085 | 1997-07-17 | ||
US08/749,164 | 1997-07-17 |
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EP (4) | EP1384403B1 (zh) |
JP (1) | JP4584361B2 (zh) |
KR (1) | KR100517028B1 (zh) |
CN (1) | CN1279976C (zh) |
AP (1) | AP9901540A0 (zh) |
AT (2) | ATE274805T1 (zh) |
AU (1) | AU744537B2 (zh) |
BR (1) | BR9712952A (zh) |
CA (1) | CA2272417C (zh) |
DE (2) | DE69737218T2 (zh) |
IL (1) | IL129919A (zh) |
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- 1997-07-17 US US08/896,085 patent/US5980898A/en not_active Expired - Lifetime
- 1997-11-14 DE DE69737218T patent/DE69737218T2/de not_active Expired - Lifetime
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- 1997-11-14 EP EP03017154A patent/EP1384403B1/en not_active Revoked
- 1997-11-14 AT AT97947608T patent/ATE274805T1/de not_active IP Right Cessation
- 1997-11-14 DE DE69730534T patent/DE69730534T2/de not_active Expired - Lifetime
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- 1997-11-14 JP JP52294298A patent/JP4584361B2/ja not_active Expired - Fee Related
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EP1014787A4 (en) | 2002-08-07 |
AU744537B2 (en) | 2002-02-28 |
NZ335749A (en) | 2001-01-26 |
JP4584361B2 (ja) | 2010-11-17 |
BR9712952A (pt) | 1999-12-07 |
CA2272417A1 (en) | 1998-05-22 |
EP1384403A1 (en) | 2004-01-28 |
DE69730534T2 (de) | 2005-09-15 |
AU5265298A (en) | 1998-06-03 |
EP2272526A3 (en) | 2012-02-08 |
EP2272526A2 (en) | 2011-01-12 |
CA2272417C (en) | 2003-07-29 |
KR20000053306A (ko) | 2000-08-25 |
DE69730534D1 (de) | 2004-10-07 |
KR100517028B1 (ko) | 2005-09-26 |
IL129919A (en) | 2008-04-13 |
US20060269593A1 (en) | 2006-11-30 |
ATE274805T1 (de) | 2004-09-15 |
EP1384403B1 (en) | 2007-01-03 |
US7037499B1 (en) | 2006-05-02 |
EP1557177A1 (en) | 2005-07-27 |
DE69737218D1 (de) | 2007-02-15 |
EP1014787A1 (en) | 2000-07-05 |
CN1279976C (zh) | 2006-10-18 |
DE69737218T2 (de) | 2007-11-08 |
EP1014787B1 (en) | 2004-09-01 |
IL129919A0 (en) | 2000-02-29 |
US5980898A (en) | 1999-11-09 |
WO1998020734A1 (en) | 1998-05-22 |
JP2001511115A (ja) | 2001-08-07 |
AP9901540A0 (en) | 1999-06-30 |
ATE349890T1 (de) | 2007-01-15 |
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