CN1086589C - 疫苗 - Google Patents
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- CN1086589C CN1086589C CN94194554A CN94194554A CN1086589C CN 1086589 C CN1086589 C CN 1086589C CN 94194554 A CN94194554 A CN 94194554A CN 94194554 A CN94194554 A CN 94194554A CN 1086589 C CN1086589 C CN 1086589C
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- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
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Abstract
本发明提供了疫苗组合物,含有一种水包油乳液,该乳液可任选地与3-脱氧酰基化单磷酰脂A和QS21相组合。该疫苗组合物是许多免疫应答的强有力的诱导剂。
Description
本发明涉及新的疫苗制剂,及其生产方法以及该疫苗制剂在医学上的应用。特别是,本发明涉及一种水包油乳液。这种乳液包括生育酚、角鲨烯、吐温80、Span 85(司盘85)以及卵磷脂,并且具有有效的佐剂特性。含有一种来源于Quillaja Saponaria Molina的树皮的HPLC纯化的非毒性组分,QS21,和/或3脱氧酰基化的单磷酰脂A(3De-O-acylated monophosphoryl lipid A,3D-MPL),以及这样的水包油乳液进行组合的疫苗也是本发明的一部分。
从GB 2220211(Ribi)知道了3脱氧酰基化的单磷酰脂A。从化学上说,它是一种具有4,5或6酰基化的链的3脱氧酰基化的单磷酰脂A的混合物,由Ribi Immunochem Montana制备。在国际专利申请NO.92/116556中公开了3脱氧酰基化的单磷酰脂A的一种优选形式。
QS21是一种来自南美的Quillaja Saponana Molina树的树皮的经Hplc纯化的皂角苷(Saponin)的非毒性组分,其生产方法公开(以QA 21形式)于美国专利NO.5,057,540中。
水包油乳液本身是现有技术中已知的,并且已有人建议可将其用作为佐剂组合物(EPO 399843)。
本发明是基于下列惊人发现,与现有技术的乳液不相同的本发明的水包油乳液,含有生育酚或者含有结合了QS21和/或3D-MPL的生育酚,从而增强了对给定抗原的免疫应答。这种增强作用比迄今为止以前的所有同类制剂能提供更好的免疫应答。
另外,当将本发明的水包油乳液与3D-MPL和QS21一起配制时是IgG2a产生和TH1细胞应答的优选的刺激剂。这是有利的,因为在细胞介导的应答中已知包含有TH1应答。的确在鼠中IgG2a的诱导是与这样一种免疫应答相关的。
例如以这种方式组合的HIV抗原gp120的疫苗制剂可强有力地增效诱导对gp 120蛋白质的特异的免疫应答。
观察到可能诱导强的细胞溶解的T淋巴细胞应答的结果很有意义,因为在一些动物模型中已经证明这种细胞应答诱导了抵抗疾病的保护作用。
本发明人已经证明,佐剂QS21和3D-MPL以及在一起的水包油乳液与一种抗原结合后在脾脏中导致对CS蛋白质特异性的CTL的强力诱导作用。QS21本身也增强了CTL的诱导,而3D-MPL不能。
当胞内合成靶抗原时(例如由病毒,胞内细菌产生的感染,或在肿瘤中)容易看到CTL的诱导作用,因为由抗原的蛋白质裂解产生的肽可以进入合适的加工途径,导致以与I类分子相结合形式呈现在细胞膜上。但是,一般来说,预形成的可溶性抗原没有到达该加工和呈现途径,并且没有诱导I类范围内的CTL。因此,可诱导抗体和T辅助应答的常规的非活性疫苗一般不能诱导CTL介导的免疫应答。两种佐剂QS21和3D-MPL与水包油乳液结合在一起能够克服基于重组蛋白质的疫苗的严重的局限性,并可诱导更宽范围的免疫应答。
已经证明在鼠模型系统中特异于CS蛋白质的CTL可抵抗疟疾(Romero等人,Nature 341:323(1989))。在人试验中,当将志愿者用恶性疟原虫的被辐射过的子孢子体进行免疫接种后,可显示出抵抗随后的疟疾的进攻,显示出诱导出了特异于CS表面抗原决定簇的CTL(Malik et al.Proc.Natl.Acad.Sci.USA 88:3300(1991))。
可诱导出对以重组分子形式给药的一种抗原具特异性的CTL的能力是与疟疾疫苗的研制相关的,因为基于免疫应答的产生及其特性,使用经辐射的子孢子体是不切实际的。
RTS是一种杂合蛋白质,它基本上含有借助于乙肝表面抗原的pre S2部分的四个氨基酸连接到乙肝病毒的表面(S)抗原上的恶性疟原虫的环子孢子体(CS)蛋白质的整个C末端部分。其完整结构公开于共同待批国际专利申请NO.PCT/EP 92/02591,该申请要求了UK专利申请NO.9124390.7的优先权,其公开号为WO 93/10152。当在酵母中表达时产生出脂蛋白颗粒形式的RTS,并且当以与HBV的S抗原共表达时产生出已知为RTS,S的混合颗粒。
除了人免疫缺陷病毒和疟疾疫苗外,诱导CTL应答的能力有利于抗单纯性疱疹病毒,巨细胞病毒以及通常的具有胞内生活期的病原体的所有病例的疫苗。
同样,将重组肿瘤抗原和两种佐剂组合后可诱导特异于已知肿肿瘤抗原的CTL。这为研制抗癌疫苗提供了条件。
在一些系统中,将3D-MPL和QS21以及水包油乳液结合起来能够协同地增强干扰素γ的产生。本发明人利用已知的名为gD2t的单纯性疱疹抗原证明了3D-MPL和QS21与水包油乳液组合在一起的潜力。gD2t是一种从HSV-2上切下的可溶性糖蛋白D,并且是采用Berman等人的方法(Science 222 524-527)在CHO细胞中产生的。
IFN-γ的分泌是与抗胞内病原体,包括寄生虫,细菌和病原体的保护性应答相关的。由IFN-γ对巨噬细胞的激活增强了胞内对微生物的杀伤力以及增强Fc受体的表达。也可能存在直接的细胞毒性,尤其是在与淋巴毒素(TH1细胞的另一种产物)进行增效作用时。IFN-γ既是NK细胞的诱导剂又是其产物,NK细胞是保护作用的主要的固有的效应子。通过IFN-γ或者其他机制进行的TH1型应答为IgG2a免疫球蛋白同型提供了优先帮助。
糖蛋白D定位于病毒包膜上,并且也存在于受感染细胞的胞质中(Eisenberg R.J.等人J.of Virol.1980 35:428-435)。它含有包括信号肽在内的393个氨基酸并且具有约60KD的分子量。在所有的HSV包膜糖蛋白中,它可能是得到最详细表征的一种(Cohen等人,J.Virology 60:157-166)。已知在体内它对于病毒粘附到细胞膜上起着关键的作用。而且,已证明糖蛋白能够在体内诱导出中和抗体(Eing等人,J.Med.Virology 127:59-65)。但是,尽管在患者血清中存在高效价的中和抗体,潜伏的HSV2病毒仍能被重新激活并诱导疾病复发。因此,显然仅具有诱导中和抗体的能力不足于控制疾病。
为了防止疾病复发,任何疫苗需要不仅刺激中和抗体,而且激发由T细胞,特别是毒性T细胞介导的细胞免疫作用。
对于gD2t例子,它是308个氨基酸的HSV2糖蛋白D,它含有天然存在的糖蛋白的第1-306个氨基酸,在该被截短的蛋白质的C-末端添加了天冬酰胺和容氨酰胺。这种蛋白质形式包括信号肽,将该信号肽裂解后产生成熟的283氨基酸蛋白质。在Genentech的欧洲专利EP-B-139 417中已描述了在中国仓鼠卵细胞中生产这样一种蛋白质。
该成熟的被截短的糖蛋白D(rgD2t)或从哺乳动物细胞中分泌的等同蛋白质优先用于本发明的疫苗制剂中。
在患有生殖器疱疹的豚鼠模型中本发明的制剂可十分有效地诱导保护性免疫作用。甚至在具有非常低剂量抗原(例如低至5μgrgD2t)时该制剂可保护豚鼠抵抗初次感染,并且也激发了特异性的中和抗体应答。本发明人利用本发明的制剂也证实了在鼠中效应细胞介导的TH1型应答。
因此,在一个优选实施方案中,本发明提供了一种含有与3-脱氧酰基化的单磷酰脂A,QS21和一种水包油乳液相组合的抗原的疫苗或药物制剂,该水包油乳液含有一种可代谢的油,例如角鲨烯,α-生育酚和吐温80。这样一种制剂适合于很多的单价或多价疫苗。另外,该水包油乳液可含有span 85。3-脱氧酰基化的单磷酰脂A的一种优选形式公开于公开号为NO.92116556的Smithkline BeechamBiologicals s.a.的国际专利申请中。
该水包油乳液可以单独使用或与其他佐剂或免疫刺激剂结合起来使用,因此本发明的一个重要实施方案是一种含有角鲨烯或另一种可代谢油,α-生育酚和吐温80的水包油制剂。该水包油乳液还可含有span 85和/或卵磷脂。
优选的疫苗制剂含有能够引发抗人或动物病原体的免疫应答的抗原或抗原组合物,所述抗原或抗原组合物来源于HIV-1(例如gp120或gp160),任何猫的免疫缺陷病毒,人或动物疱疹病毒,例如gD或其衍生物或立即早期蛋白如来源于HSV1或HSV2的ICP27,巨细胞病毒((尤其是人)(例如gB或其衍生物),水痘带状疱疹病毒(例如gpI,II或III),或者来源于例如已肝病毒的肝炎病毒,例如乙肝表面抗原或其衍生物,甲肝病毒,丙肝病毒和戊型肝炎病毒,或者来自于其他病毒病原体,例如呼吸道合胞体病毒,人乳头瘤病毒或流感病毒、或者来源于细菌病原体如沙门氏菌、奈瑟氏菌、螺旋体(例如OspA或OspB或其衍生物),或者衣原体,或博德特氏菌例如P.69,PT和FHA,或来源于寄生虫如疟原虫或者弓形体。
该制剂还可含有一种抗肿瘤抗原并适用于采用免疫治疗法治疗癌症。
在一种适于免疫治疗的患有B细胞淋巴瘤的动物模型中,当在第0天时将BCL-1鼠淋巴瘤细胞静脉注射到Balb/c小鼠中,并在第3天,10天和20天用BCL-1同种异型给小鼠免疫接种,就抗体效价和存活率(该组为存活率为100%的唯一组)而言,SB62/MPL/QS21制剂被证明为最有效力。同样,该制激发针对所包含的抗原的细胞毒性T淋巴细胞的能力,使其成为作为癌抗原制剂的良好的候选物(例如用于通过活性免疫接种而对肿瘤进行免疫治疗的黑色素瘤抗原MAGE-1和MAGE-3)。
该制剂还适用于与疱疹的轻颗粒一起使用,例如描述于国际专利申请NO.PCT/GB 92/00824,以及国际专利申请NO.PCT/GB 92/00179的制剂。
乙肝表面抗原的衍生物是本领域内熟知的,并且特别包括那些在欧洲专利申请EP-A-414374;EP-A-0304578,和EP 198-474中描述的PreS1,PreS2 S抗原。在一个优选的方面,本发明的疫苗制剂包括HIV-1抗原,gp120,尤其是当在CHO细胞中表达时,在另一实施方案中,本发明的疫苗制剂包括如上文中定义的gD2t。
在本发明的又一方面,提供了如本发描述的在医学上使用的疫苗。
QS21与3D-MPL的比例通常是在1∶10至10∶1的范围,优选的是1∶5-5∶1并且实质上经常是1∶1。达到最适增效作用的优选范围是2.5∶1到1∶1的3D MPL∶QS21。当给人使用时通常疫苗中存在的QS21与3D MPL的比例为1μg-100μg/每剂量的范围,优选的是10μg-50μg/每剂量。水包油乳液通常含有2-10%的角鲨烯,2-10%α-生育酚以及0.3-3%吐温80。优选的是角鲨烯与α-生育酚的比例是等于或者低于1,因为这样使乳液更稳定。还可以存在1%含量的Span 85。在某些情况下,本发明的疫苗进一步含有一种稳定剂是有利的。
通常疫苗的制备可参见New Trends and Developments in Va-ccines,edited by Voller et al.,University Park Press,Baltimore,Maryland,U.S.A.1978。脂质体内进行包埋的方法例如可参见Fullerton,美国专利4,235,877的描述。蛋白质与大分子的结合例如公开于由Likhite的美国专利4,372,945以及Armor等人的美国专利4,474,757。
所选择单疫苗剂量中蛋白质的量为在典型的接种人中诱导产生免疫保护应答而无显著的毒副作用所需的量。该量将随所使用的特定免疫原以及如何提供而变化。通常,单个剂量含有1-1000μg的蛋白质,优选的是1-100μg,更优选的为4-40μg。采用包括观察个体中合适免疫应答的标准研究确定用于特定疫苗中的最适量。在首次免疫接种之后,受试个体可接受一次或者适当间隔的几次加强免疫接种。
本发明的药剂可用于预防和治疗目的。
因此,一方面,本发明提供了一种治疗方法,该方法包括给患者施用有效量的本发明的疫苗。
下面实施例阐明本发明。
实施例1
含有HIV-1的gp 120抗原的疫苗制剂
制备两种佐剂制剂,每种含有下列水包油乳剂成分。
SB 26:5%角鲨烯,5%生育酚,0.4%吐温80;颗粒大小是500nm。
SB 62:5%角鲨烯,5%生育酚,2.0%吐温80;颗粒大小是180nm。
1(a)乳剂SB 62的制备(2倍浓缩)
将吐温80溶于磷酸缓冲盐水(PBS)中得到溶于PBS中的2%的溶液。为了提供100ml两倍浓缩乳液,将5克DLα生育酚和5ml角鲨烯涡流以便彻底混合。加入90ml PBS/吐温溶液并彻底混合。然后将得到的乳液通过一个注射器并且最后利用MllOS微射流机器进行微射流。得到的油滴具有约180nm的大小。
1(b)乳液SB26的制备
以类似的方式利用0.4%吐温80制备该乳液。
1(c)以类似的方式制备表1中描述的其他乳液。下列实施例中详细描述了检测这些乳液的试验。
1(d)gp120 QS21/3D MPL水包油制剂的制备
向1a)或b)或c)乳液中加入某体积的两倍浓缩的rgp 120(20μg或100μg)并且混合。将它与50μg/ml的3D-MPL和20μg/ml的QS21合并,得到最后的制剂。按照盐含量和pH值调整缓冲液。
表3显示利用来自HIV的gp120和50μg/ml 3D-MPL(MPL)以及20μg/ml的QS21的SB26的效果。结果显示在第二次(PII)和第三次(PIII)接种后效价的几何平均值(GMT),以及对淋巴细胞增殖和γ干扰素产生作出的细胞介导的应答(CMI)。
实施例2
引言:一种HIV gp120乳液系统的评估
在该实验中,对四个乳液[SB26,SB62,SB40,SB61]进行比较。对各个制剂成分(抗原,乳液,3D-MPL,QS21)的效应进行评估。
2(b)所使用的动物试验组
使用22组,每组5个动物,使每组接受不同的疫苗制剂。
-1-4组:gp120(10μg)/无乳液±[3D-MPL,QS21]
-5-9组:gp120(10μg)/SB26±[3D-MPL,QS21]
-10组:无抗原/SB26+[3D-MPL,QS21]
-11-12组:gp120(10μg)/SB62±[3D-MPL,QS21]
-13-16组:gp120(10μg)/SB40±[3D-MPL,QS21]
-17-20组:gp120(10μg)/SB61±[3D-MPL,QS21]
-21-22组:gp120(5μg)/SB26±[3D-MPL,QS21]
-分析:抗gp120W61D的抗体效价以及同型分析(所有组)
2(c)免疫接种以及抽血时间表
-用gp120W61D对动物进行免疫接种,所述gp120W61D是配制于每剂量存在5μg 3D-MPL和5μg QS21的不同的油水乳液中。阴性对照接受没有抗原的等同制剂。
-在0和14天时皮下注射免疫接种动物。所给药的每次注射剂量为100μl。
-在免疫接种前(0天)以及在免疫接种之后第14天(第一次接种后),第21以及28(第二次接种后第7和第14天)天获取血样品。
2(d)血清学应答分析:
-第一次接种后和第二次接种后的14天的血清学应答是采用针对gp120W61D的直接ELISA检测法进行评估的。
-用免疫接种之后小鼠中诱导出的gp120W61D特异性抗体的同型也对第二次接种后14天的应答进行表征。
3.结果和讨论
结果描绘于表2中。
a)在存在或不存在3D-MPL/QS21的乳液的比较:
-将SB26,SB40或SB62乳液加入到抗原中诱导出更高的抗体效价。在没有免疫刺激剂情况下,gp120特异性抗体基本上是IgG1。
-加入免疫刺激剂3D-MPL和QS21诱导了巨大的血清学应答并且抗体从IgG1型转变为IgG2a/IgG2b:这与细胞介导的免疫相关。
优选的组合是[SB26+MPL+QS21]。
c)gp120/SB26制剂:
在8组和9组之间没有观察到血清学应答之间的显著差异:在本制剂其他成分之前或之后加入gp120。
d)抗原剂量:
配制于SB26中的5和10μg gp120诱导了高血清应答(5-8组以及21-22组)。
实施例3
HSV rgD2t制剂
按照实施例1(a)中提出的类似方式,制备含有单纯疱疹抗原rgD2t的制剂并用于免疫接种豚鼠。所述制剂诱导出抵抗豚鼠模型中复发和起始疾病的保护作用。
实施例4
利用个体基因型作为免疫原筛查用于诱导保护性抗淋巴瘤应答的佐剂。
利用来自BCL1淋巴瘤细胞的个体基因型给Balb/c小鼠进行治疗性免疫接种。
由Yefenoh等人对BALB/C B细胞淋巴瘤模型进行了综述。Curr-ent opinions Immunobiology 1993 5:740-744。
用104肿瘤细胞在0天时注射(ip)的10只小鼠的组,并用溶于不同佐剂制剂中的100μg的KLH偶合的抗BCL1抗原决定簇的免疫球蛋白(KLH/Ig:1/1的比例)在第3天,10天,20天免疫接种(在背部皮下注射)。对抗KLH和抗个体基因型的血清抗体水平以及鼠死亡率进行了监测。
所检测的制剂:
组号 佐剂 MPL:10μg
1 无(无抗原) QS21:10μg
2 无
3 弗氏佐剂
4 明矾
5 明矾/MPL
6 明矾/MPL/QS21
7 QS21
8 MPL/QS21
9 SB62MPL
10 SB62/MPL/QS21
12-15组:没有抗原的不同佐剂
与其他组相比,制剂8、9、10的行为均较好。在抗体效价以及存活率两方面,制剂10表现为最有效力(生存率为100%的唯一组)。
实施例5
RTS,S的不同制剂
a)在猴中的评价
在国际专利申请No.WO 93/10152中描述了RTS,S并且制成制剂用于给Rheusus免疫接种。每组有5个动物:
I组 RTS,S,3D-MPL(50μ),Al(OH)3
II组 RTS,S,QS21(20μ),Al(OH)3
III组 RTS,S,3D-MPL(50μ),QS21(20μ)
IV组 RTS,S,3D-MPL(50μ),QS21 Al(OH)3
V组 RTS,S,3D-MPL(10μ),QS21 Al(OH)3
VI组 RTS,S,3D-MPL(50μ),QS21 SB60
给动物接种并且在第一次免疫接种之后14天以及第二次免疫接种之后12天取血,并检测其抗乙肝表面免疫球蛋白。如在图1中可看到的,接受SB60中的RTS,S的动物具有的抗体效价几乎比任何其他组高出6倍。
b)用小鼠对RTS,S的各种制剂进行评估
7组动物接受下列制剂
1组 RTS,S SB26
2组 RTS,S QS21 3D-MPL
3组 RTS,S QS21 3D-MPL SB62
4组 RTS,S 3D-MPL Al(OH)3
5组 RTS,S Al(OH)3
6组 空白
7组 阴性对照
(RTS,S-5μg/剂量,3D-MPL 5μg/剂量,QS21 5μg/剂量)
给动物接种并且在第一次接种后15天以及在第二次接种之后7天和15天取血,并检测抗HBSAg抗体亚型。如在图2中可看到的,用QS21和3D-MPL配制的乳液SB62优先地和以协同增效方式加强IgG2a抗体应答,而单独的SB62或3D-MPL/QS21诱导极少的IgG2a应答。
实施例6:对不同的B burgdorferi OspA制剂的评估
6.1不同的B burgdorferi ZS7 OspA脂蛋白制剂的评估
在欧洲专利申请0418 827(Max Plank et al)中描述了B bur-gdorferi的OspA脂蛋白。
在balb/c鼠中检测下列制剂
1. OspA+Al(OH)3
2. OspA+Al(OH)3+3D-MPL(10μ)
3. OspA+Al(OH)3+3D-MPL(30μ)
4. OspA+Al(OH)3+3D-MPL(10μ)+QS21(5μ)
5. OspA+Al(OH)3+3D-MPL(30μ)+QS21(15μ)
6. OspA+SB60+3D-MPL(10μ)+QS21(5μ)
7. OspA+SB60+3D-MPL(30μ)+QS21(15μ)并且在第一次接种之后7天以及在第二次接种之后7天(在0天和14天接种)研究抗体效价和亚型。
在图3和图4中以图形式描述结果,并且证明本发明的制剂诱导出高水平的抗体并且它们优先的是IgG2a亚型。
实施例7
a)HSV-2 ICP27
在0天和14天时在后脚爪用各种NS1-ICP27制剂给雌性Balb/c小鼠免疫接种。每次注射含有5μg的NS1-ICP27以及SB26水包油乳液,QS21(10μg)和MPL(25μg)的组合物。在28天时取出腘淋巴结细胞并在体外用转染了ICP27基因的同系P815细胞进行刺激。用转染了ICP27和P815 ICP27阴性对照的P815靶细胞检测该培养物的特定的细胞溶解活性。
对于不同的免疫接种组以不同的效应子∶靶(E∶T)比例的具体裂解结果如下所示:ICP 27(5μg)E∶T P815 转染了ICP27克隆121的P815100∶1 -1 030∶1 -2 -310∶1 3 03∶1 1 01∶1 2 20.3∶1 2 2ICP 27(5μg)+MPL(25μg)E∶T P815 转染了ICP27克隆121的P815100∶1 5 730∶1 2 210∶1 1 23∶1 -1 -11∶1 -2 -20.3∶1 -4 -1ICP 27(5μg)+QS21(10μg)E∶T P815 转染了ICP27克隆121的P815100∶1 4 1730∶1 5 1010∶1 3 73∶1 4 51∶1 3 50.3∶1 0 1ICP 27(5μg)+SB26E∶T P815 转染了ICP27克隆121的P815100∶1 5 2030∶1 1 1910∶1 2 123∶1 -2 71∶1 1 50.3∶1 1 2ICP 27(5μg)+MPL(25μg)+QS21(10μg)E∶T P815 转染了ICP27克隆121的P815100∶1 4 1330∶1 5 1210∶1 4 173∶1 1 31∶1 0 30.3∶1 -1 -2ICP 27(5μg)+MPL(25μg)+QS21(10μg)+SB26E∶T P815 转染了ICP27克隆121的P815100∶1 2 2030∶1 0 1710∶1 3 193∶1 3 81∶1 1 60.3∶1 2 3在免疫接种组中获得的低ICP27特异性裂解%ICP27(5μg)+QS21(10μg)ICP27(5μg)+SB26ICP27(5μg)+MPL(25μg)+QS21(10μg)ICP27(5μg)+MPL(25μg)+QS21(10μg)+SB26而ICP27(5μg)ICP27(5μg)+MPL(25μg)是阴性
因此这些数据显示由重组NS1-ICP27在单独的水包油乳液或与QS21和MPL一起;或与QS21一起诱导出CTL。
b)用不同疫苗(NS1-ICP27/NS1-ICP27 MPL+QS21/NS1-ICP27 SB26=MPL和QS21/仅佐剂)在脚爪处给5个Balb/c小鼠为一组的各组免疫接种。一个剂量含有10μg NS1-ICP27,10μg MPL和10μg QS21。在0天和7天进行两次免疫接种。在14天用5.2×103TCID50的HSV2菌株MS攻击小鼠。一直到攻击后14天都记录是否出现带状疱疹样的损伤和死亡。
在大肠杆菌中表达出了与流感病毒NS1片段的融合蛋白形式的HSV2的ICP27。在鼠带状疱疹样模型中对该纯化重组蛋白与MPLQS21制剂的组合物的保护效率进行评估。在用与MPL+QS21或与水包油乳液(SB26)+MPL和QS21组合的NS1-ICP27进行两次免疫接种的Balb/c小鼠可完全抵抗疾病(无带状疱疹样损伤)以及在HSV2野生型攻击之后没有死亡发出。相反,在单独用NS1-ICP27或用NS1-ICP27与SB26但没有MPL和QS21进行相组合的疫苗对小鼠进行免疫接种后没有观察到保护作用。
表1
两倍浓缩的载体
乳液SB | 生育酚% | 角鲨烯% | 吐温80% | Span 85% | 卵磷脂% | 大小 |
2626.163646162 | 555555 | 555555 | 0.40.40.60.812 | 000000 | 00.10000 | 500nm 90-100%800nm 10-0%500nm500nm500nm250-300nm180nm |
4040.1606566 | 55555 | 55555 | 0.40.410.40.4 | 1111.52 | 00.1000 | 500nm 80-100%800nm 20-0%500nm300nm500nm500nm |
表2
HIV gp 120W61D/ 鼠免疫原性(94243)/BALB/C(F.P.)
组号 | 免疫原(剂量)/制剂 | ELISA效价(第二次接种后7天) | %IgG1 | %IgG2a | %IgG2b |
123456789111213141516171819202122 | gP120 10μggP120 10μg+3D-MPL 5μggP120 10μg+QS21 5μggP120 10μg+3D-MPL+QS21gP120 10μg/SB26gP120 10μg/SB26+3D-MPLgP120 10μg/SB26+QS21gP120 10μg/SB26+3D-MPL+QS21SB26+3D-MPL+QS21/gP120 10μggP120 10μg/SB62gP120 10μg/SB62+3D-MPL+QS21gP120 10μg/SB40gP120 10μg/SB40+3D-MPLgP120 10μg/SB40+QS21gP120 10μg/SB40+3D-MPL+QS21gP120 10μg/SB61gP120 10μg/SB61+3D-MPLgP120 10μg/SB61+QS21gP120 10μg/SB61+3D-MPL+QS21gP120 5μg/SB26gP120 5μg/SB26+3D-MPL+QS21 | 4944164215155274912205873885102017816918570410348217393632028521948953209217<5077515407375967325089242736 | 1005489229431732322925490317814-3174299918 | 0154602421557608377441567-501357061 | 03281842713211909425718-191314121 |
由LINEST计算出的gp120W61D的ELISA效价:5个单体效价的几何平均值
表3
基于3D-MPL的制剂;HIV工程猴研究
读数 | GMT ELisa W61 D | GMT Neut.MN | 体内DTH | 体外CMI | ||||
制剂 | P11 | P111 | P11 | P111 | LP | IL-2 | γIFN | |
gp120(100μg)/油/水+MPL+QS21gp120(20μg)/油/水+MPL+QS21豚鼠中“过去的”gp120(100μg)/油/水+MPL | 6052352026 | 934105015020064 | 1∶5001∶500 | >1∶32001∶2400 | ++ | 没有检测没有检测 | ++ |
Claims (10)
1、一种疫苗组合物,包含有一种抗原和/或抗原组合物,QS21,3脱氧酰基化单磷酰脂A(3D-MPL)和水包油乳液,其中该水包油乳液具有下列组分:一种可代谢油,例如角鲨烯,α-生育酚和吐温80。
2、按照权利要求1中所述疫苗,其中QS21∶3D-MPL的比例是从1∶10到10∶1。
3、按照权利要求1或2所述疫苗组合物,能够在哺乳动物中激发针对该抗原或抗原组合物的细胞溶解性T细胞应答。
4、按照权利要求1-3任一项所述的疫苗组合物,它能够刺激干扰素γ的产生。
5、按照权利要求1-4任一项所述疫苗组合物,其中QS21∶3D-MPL的比例是从1∶1到1∶2.5。
6、如本文所述的一种疫苗组合物,其中包括来源于人免疫缺陷病毒、猫免疫缺陷病毒、单纯性疱疹病毒1型、单纯性疱疹病毒2型、人巨细胞病毒、甲、乙、丙或戊型肝炎病毒、呼吸道合胞体病毒、人乳头瘤病毒、流感病毒、沙门氏菌、萘瑟氏菌、螺旋体、衣原体、博德持氏菌、疟原虫或弓形体中的任何抗原或抗原组合物。
7、根据权利要求1-5任一项所述的疫苗,其中该抗原是肿瘤抗原。
8、权利要求1-5任一项所述组合物的应用,用于制造病毒,细菌或寄生虫感染的预防治疗的疫苗。
9、权利要求1-5任一项所述组合物的应用,用于制造病毒,细菌或寄生虫感染或癌症的免疫治疗的疫苗。
10、一种制备权利要求1-5所述疫苗组合物的方法,包括将QS21,3D-MPL和如权利要求1中定义的水包油乳液与一种抗原或抗原组合物进行混合。
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