CN1185740A - 衣原体疫苗 - Google Patents
衣原体疫苗 Download PDFInfo
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- CN1185740A CN1185740A CN96194300A CN96194300A CN1185740A CN 1185740 A CN1185740 A CN 1185740A CN 96194300 A CN96194300 A CN 96194300A CN 96194300 A CN96194300 A CN 96194300A CN 1185740 A CN1185740 A CN 1185740A
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Abstract
本发明提供了一种新的利用外膜蛋白和两个免疫刺激因子即3D-MPL和QS21预防衣原体感染的疫苗。
Description
本发明涉及一种抗衣原体感染的疫苗配方,尤其涉及一种含有重组MOMP且以QS21和3D-MPL为佐剂的配方。
专性的胞内细菌沙眼衣原体感染结膜和泌尿生殖道的粘膜上皮细胞,造成多种人类疾患如沙眼和生殖器的各种感染,而后者能够导致长期的后遗症。沙眼流行于若干个发展中国家,是引起可预防性失明的首要原因;生殖器感染的病例在美国每年约有三百万起,其能使每年有200,000个妇女因衣原体感染的输卵管炎而丧失生育能力(1)。因此,这种病原体是影响公众健康的重大问题,致力于建立一种抗人类衣原体感染的疫苗的努力就显得十分必要。
在人类和非人类灵长类动物身上的疫苗实验表明,以完整的生物体作为免疫原能产生血清种-特异的保护,但是有些这样的疫苗在再次感染时往往产生严重的反应(2)。一些研究证明,衣原体感染从病理学角度上讲是受免疫介导的(3)。此外,纯化的衣原体57KDa(Hsp60)会引起和事先感染的动物的再次感染相似的病理特征(4,5)。基于以上的发现可以得出这样的结论:仅使用一种亚单位疫苗就可以达到预防沙眼衣原体感染的目的。
沙眼衣原体的种类可分成15个血清型,它们又分属于3个血清群:B复合体(包括血清型B、Ba、D、E、L1和L2),中间复合体(血清型F、G、K和L3)和C复合体(血清型A、C、H、I和J)(6)。性病(STD)是由横跨3个血清群的血清型D到K引起。因此,抗衣原体性病(STD)的亚单位疫苗应当能够抵抗多种血清型(它们或多或少有一定抗原相关性)。
在设计亚单位疫苗的时候,更多的兴趣是集中在存在于一种40KDa的主要外膜蛋白(MOMP)中的血清型抗原上。这种外膜蛋白在体外实验中有孔素(porin)功能(7),存在于细菌的整个生命周期中(8);该主要的表面蛋白在人和动物体内具有极高的免疫原性。该MOMP有4个可变区(VD),外围由五个在各种血清型中高度保守的恒定区包围(9,10)。在体内和体外,中和B细胞的抗原决定簇已被定位在可变区(VD)(11,12,13,14,15)上,而T细胞的抗原决定簇既可位于可变区也可位于恒定区(16,17)。已有不同的科学家将重组的外膜蛋白(MOMP)在大肠杆菌中表达(19,20)。但是manning等人指出他们获得的重组蛋白不能和识别一种构象MOMP抗原决定簇的单抗反应(18)。
在多种动物模型中,用重组的或纯化的MOMP免疫动物,接着用同型或异型衣原体攻击动物,结果对感染参数有不同的影响(21,22,23)。在小鼠上建立了一个非常好的输卵管炎的实验模型。在此模型里,在小鼠子宫内接种人类沙眼衣原体导致了长期不孕(24,25)。在异型攻击实验中,Tuffrey等人发现用铝胶吸附的rMOMP非肠道和粘膜免疫分别降低了输卵管炎的严重性和下生殖道移地发育的持续时间。但是,该制剂对感染引起不育没有保护作用(23)。
细胞免疫和体液免疫似乎对沙眼衣原体造成的生殖器病变均有保护意义。但是,Rank研究小组指出在小鼠体内T细胞介导的免疫是控制衣原体生殖器疾病的主要免疫机制(26,27,28),而且也发现CD4和CD8阳性的T细胞在体内对抗衣原体的免疫反应有作用(29,30)。
3脱-O-酰基单磷酰类脂A见于GB2 220 221(Ribi)。从化学组成上讲,它是由3-脱酰基单磷酰类脂A和4,5或6酰基化链的混合物且是由Ribi免疫化学蒙大拿公司制造。
QS21是由HPLC纯化的皂苷的非毒性组分。这种皂苷来源于南美Quillaja saponaria molina树的皮,它的提取纯化方法见于美国专利No.5,057,540。包含QS21和3D-MPL的疫苗公开于国际专利申请No.WO94/PP153。
本发明首次提供了一种能有效地预防衣原体感染所致的不孕的疫苗组合物。
相应地,本发明提供了包括3D-MPL,QS21和来源于衣原体的MOMP的疫苗配方。特别地,该疫苗含有来源于B复合体血清群的衣原体的MOMP。优选地,该疫苗至少含有一种来源于L2血清型的MOMP,但还可以含有来源于其他血清型如D和E的抗原。
在一个优选的实施例中,QS21和甾醇一起提供。研究表明这样的组合物不仅降低了反应原性而且提高了QS21对碱介导的水解的稳定性。
在本发明优选的组合物中,QS21和包括胆固醇在内的脂质体结构相结合(此后称为DQ)。这种佐剂组合物在同时待审的英国专利申请9508326.7和9513107.4中有详尽描述,这些申请的公开内容以参考文献的形式并入本发明。在一个优选的配方中,抗原和MPL在脂质体结构之外。
疫苗制备一般见于Voller等人编著的《疫苗的新趋势和新进展》中(Park大学出版社,巴尔的摩,Maryland,美国,1978年)。脂质体中微囊化见于诸如Fullerton的美国专利4,235,877。蛋白质和大分子结合公开于诸如Likhite的美国专利4,472,945和Armor等人的美国专利4,474,757。
每一疫苗剂量中蛋白质的量选择为在典型疫苗中可诱发起免疫保护反应但却不引起明显的副作用的量。这个量因所用的特定免疫原及其存在形式而变。通常每剂疫苗中有1~1000μg的蛋白,优选地为2~100μg,最优选地是4~40μg。每种具体疫苗的最佳用量可由标准的研究方法(包括观察实验对象的合适免疫反应)而得以确定。初次接种以后,实验对象可能还需要在有足够时间间隔的情况下接受一到多次加强免疫。
本发明所提供的配方既可用于预防疾病也可用于治疗目的。
从而另一方面,本发明提供了一个治疗方法,其包括对患者给于有效剂量的本发明的疫苗。
在本发明的一个实施例中,MOMP抗原从L2血清型(Serovart 2)中来并用DNA重组技术在E.Coli中生产。这种情况下,生产出的蛋白质不带有它的信号序列。
在这个发明中,抗原是否以MPL+QS21为佐剂强烈地影响了免疫实验组中IgG1∶IgG2a的比例;抗原接种时加入MPL+QS21产生低的IgG1∶IgG2a比例,而不加MPL和QS21的抗原免疫导致高的IgG1∶IgG2a比例;前者有部分保护活性而后者没有任何保护活性。有趣的是:B细胞转向生产IgG2a抗体是受γ-干扰素的介导,γ干扰素由辅助T淋巴细胞的Th1亚群产生;而IgG1的生产受Th2细胞分泌的白细胞介素4调节(40)。因为IgG2a的产生由Th1细胞产物控制,所以该保护实验组是以MPL+QS21激活了Th1细胞。在人和小鼠的模型中,Th1细胞因子,白介素2(IL-2)和γ-干扰素通常与抵抗胞内病原体感染相关,而Th2细胞因子,IL-4和IL-10,则是同渐进性疾病相关。这种情况可由小鼠对Leishmania major的近交株的耐受或敏感状况和特异性的Th1或Th2反应的诱发密切相关而得到证实(41)。此外,γ干扰素在体外有抗衣原体活性,并且在体内也参与了感染的消除(42,43)。因此,免疫刺激因子激活了Th1细胞可以说明为什么在上述的疫苗实验中观察到保护活性。
还有其它两个观察结果支持该在rMOMP+MPL+QS21免疫接种的实验组中带有保护性的细胞免疫的假说:第一,阴道分泌物中没有特异的分泌型IgA和强烈seric抗体反应的非中种特性排除了体液免疫保护的可能性;第二,应用两种不同的在MOMP VSs序列水平上差异极大的血清型获得了保护作用的异型特征,这提示来源于MOMP上加工过的保守区的T细胞抗原决定簇可能是预防不育症的因子。
以下举例说明本发明。I.材料和方法
1.1衣原体品系和实验动物
沙眼衣原体F血清型NIl品系由Tuffrey等人分离,蒙J.Orfila博士馈赠,本研究应用的是其和沙眼衣原体L2血清型434品系(ATCC,Rockville Md)。衣原体以大约106IFU/ml(包涵体形成单位/毫升)的浓度接种到MEM中的McCoy细胞上(ATCC,Rockville Md),再补充10%胎牛血清(FCS)(Gibco BRL公司)。离心1小时(1500g)并在37℃(5%CO2)下培养2小时后,移去接种物,细胞在补充了0.5μg/ml放线菌素酮(Sigma公司产品)的新鲜培养液中培养。37℃下培养48小时之后,细胞用玻璃珠裂解,收集裂解物到250mM蔗糖,10mM磷酸钠,5mM L-谷氨酸PH7.2(SPG)中,并使得浓度达到107~108IFU/ml,然后-70℃贮存。
6~8周龄的雌性C3H/HeOuJ(H-2K)小鼠从Iffa Gredo(法国)公司购得。用8~10周龄的相同品系(B&K,U.K.)的雄鼠来杂交。
1.2 PCR扩增和质粒购建
扩增:MOMPL2血清型DNA是通过在240μl裂解缓冲液中裂解10μl衣原体接种物而获得的,而PCR扩增如Denamnr等人(33)以前的报道进行。人工合成的寡核苷酸引物5’-GAGACTCCCATGGATCCACTGCCCTGTGGGGGAATCCTGC-3’和5'-TTAGAAGCGGAATTGTGCATTTAC-3'(SB Bioloyical公司,比利时)选自已经发表的序列(34)。该5'端寡核苷酸引物包含有编码成熟的MOMP分子氨基端蛋白的序列(下划线),在它之前是内切酶BamHI的识别位点(黑体)。PCR反应用Statagene公司的pfu DNA聚合酶和Kock Light NBS热循环仪(New Brunswick公司)进行。正确大小的PCR产物是用Geneclean II试剂盒(Bio lol)从1%琼脂糖凝胶上纯化的。
1.3克隆化
将扩增的L2血清型MOMPDNA用NDA聚合酶I的Klenow片段进行平端化,然后连接到已由SmaI消化的载体PGEM 4Z(promega公司)中并使用常规的CaCl2法转化入大肠杆菌JM 109中。限制性酶切分析所得的克隆,正确的DNA构建结构被增殖并且用核键(nucleobond)PC-100试剂盒(Macherey-Nagel公司)进行纯化。然后,用BamHI从PGEM4Z-MOMP上消化切出MOMP DNA,接着插入经Bam HI消化的PET15(Novagen公司)的T7lac启动子和His标志序列的下游。正确的克隆是在转化入大肠杆菌DH10B株(Stratagene公司)后用限制性酶切分析进行筛选的。克隆化的DNA的全部序列是通过双脱氧链终止法鉴定的(35)。PET15-MOMP质粒的制备是为了最终用于转化BL21(DE3)(Novagen公司),其在含有IPTG诱导的T7聚合酶时能够促进该重组产物的表达。
1.4免疫原生产、鉴定、纯化和配方
1.4.1生产和鉴定。将PET15-MOMP转化的BL21(DE3)细菌在含有200μg/ml氨苄青霉素的LB培养液中培养。当培养液的OD600约为0.6~0.8时,加入1mM IPTG以诱导表达。在诱导3小时后收集细胞,用PBS洗3遍并用含2%SDS和5%巯基乙醇的样品缓冲液裂解。在95℃下加热样品3分钟并在12%SDS-PAGE上(用在同样凝胶(Gibco BRL)上分离的分子量标记)分离总蛋白。进行免疫印迹时,将该12%SDS-PAGE分离的蛋白由胶上转移到硝酸纤维素膜上,然后用H.Caldwell博士惠赠的单抗L2 I-45和L2 I-10以及羊抗MOMP抗血清(Chemicon公司)检测。该rMOMP在细胞中的定位用Maniatis等人报道的细胞分级分离方法测定(36)。将该沉淀和上清液在样品缓冲液中重悬或调整并象总细胞裂解物一样进行分析。
1.4.2纯化。将100ml,IPTG诱导3小时的培养液用溶菌酶和脱氧胆酸盐裂解(Marston等)(37)。12,000g离心15分钟,沉淀在2ml SDS-PAGE样品缓冲液(含2%SDS,无巯基乙醇)中重悬,煮沸3分钟。再离心(1,2000g)15分钟。弃沉淀,上清补充调节到最终含Tris-HCl(PH7.9)20mM,SDS 0.5%,NaCl 500mM,咪唑5mM。然后上样到含2ml组氨酸结合树脂(Novagen)的色谱柱上。然后,按照制造商建议的方法进行金属离子亲和层析。洗脱下的产品的鉴定和纯度是用还原条件下的SDS-PAGE结合考马斯蓝染色和免疫印迹(见上述)而进行测评的。蛋白质浓度用Lowry分析法确定。含有rMOMP的部分收集在一起,用pierce公司Slide Alyser Cassettes对PBS透析过夜。
抗原配方。试用了3种配方:
1)MOMP+QS21+3DMPL
2)MOMP+SB62水包油乳浊液
3)MOMP,SB62,QS21,3DMPL
实验依WO 95/17210和/或WO 94/00153中的步骤进行。基本纯化和透析后的rMOMP用PBS稀释到25μg/ml(200μl),再和5μg MPL加10μg QS21一起注射动物。或rMOMP用PBS稀释到50μg/ml(100μl),再同100μl水包油的乳浊液(指SB62)混合,再加或不加5μgMOL/10μg QS21注射动物。对于任一种配方,疫苗制备方法均如下述:
1)MPL/QS21是通过将10nm颗粒状MPL加到MOMP抗原中,再加缓冲溶液,最后加QS21而配制。
2)MPL/QS21/SB62是通过先将抗原加到缓冲溶液,接着加SB62,再加100nm颗粒状的MPL,最后加QS21而配制。在此配方中,可以确保抗原在乳化液滴之外,MPL和大多数QS21也在其外。
3)SB62是通过将SB62加到抗原溶液中而制备。抗原在乳化液滴外。
1.5小鼠输卵管炎模型的接种,生育力及血清学随访。
每组10只雌性C3H/He OuJ小鼠在尾基部皮下注射免疫。在0周和第二周分别用200μl的含2×5μgrMOMP的上述不同配方注射,对照组则按相同时间注射含有5μgMPL和10μg QS21的乳浊液。在第六周以Tuffrey等人报到的方法接种(24)。简单地说,小鼠在攻击前7天皮下注射2.5mg黄体酮(Depo-Provera,Upjohn),而攻击是通过在双侧子宫内接种100μlSPG或McCoy细胞提取物(内含沙眼衣原体5×105 IFU)而进行的。
在第10周,处理过的雌小鼠和雄小鼠一起笼养3个月进行生育评估(1只雄鼠配两只雌鼠,雄鼠每周在各组中轮转养)。交配期之后要计算的参数是每组平均产仔数(M)和平均同窝仔大小(N)。
第六周时取血,血清用以分析rMOMP特异的抗体、在体外感染中F血清型NI1品系衣原体包涵体的异源认别和中和。在第六周收集阴道洗液,方法是取50μl PBS吸出和冲入阴道几次。而后分析rMOMP特异的分泌型IgA抗体。
1.6血清学分析
1.61抗rMOMP抗体的测定。rMOMP特异的IgG或IgA滴度通过以rMOMP为抗原的ELISA(酶联免疫吸附分析)测定。微孔板(Maxisorp,Nane)用溶于10mM碳酸盐/碳酸氢盐缓冲液(PH9.6)的5μg/ml抗原溶液4℃包被过夜,而后用洗涤液(0.1%Tween 20,PBS)洗涤,3%BSA(PBS)(Sigma产品)37℃封闭1小时。待检血清在37℃用含0.5%BSA的洗涤缓冲液(卵育液)系列稀释1小时。洗板,再加过氧化氢酶标记的羊抗鼠IgG或IgA(Sigma),37℃培育1小时。洗涤后,加反应底物邻苯二按,室温20分钟,滴加2M硫酸终止反应并在492nm波长下用Labsystems Multiskan分光光度仪测光吸收值。抗rMOMP的IgG或IgA的滴度用显示中点光吸收值的血清样品的稀释倍数的倒数表示。
对每一个血清样,该rMOMP特异的IgG反应可以用以上描述的直接ELISA稍加修改来测定rMOMP特异的IgG中IgG2b、IgG2a和IgG1的比例而仔细研究。待检血清一式三份进行培育,洗涤并分别加入用培育缓冲液稀释的生物素标记的羊抗鼠IgG2a,IgG2b,IgG1(Amersham公司)到一式三份的每一孔中。37℃培育1小时后,洗涤,再用链霉抗生物素蛋白辣根过氧化氢酶复合物(Amersham)培育1小时。显色和效价测定按上述进行。3种IgG亚型中的每一个的普遍率是以其在3个亚型的IgG的总量中所占百分比例来计算的。
1.6.2衣原体包涵体的异型检出
Mac Coy细胞在无菌的平底96孔板(Nunc公司)中培养,铺满的单层细胞用大约5×104IFU衣原体(血清种F品系NI1)感染。24小时之后,用PBS洗细胞并用甲醇固定10分钟。重复洗涤,与100μl经PBS 100倍稀释的血清样品在37℃下共培养1小时。洗涤96孔板并用过氧化氢酶标记的羊抗鼠IgG(Sigma公司产品)37℃处理1小时。PBS洗涤,加DAB(二氨基联苯胺四氢氯化物)(Sigma公司)显色。用倒置光学显微镜观察经抗rMOMP IgG显示出的NI1包涵体的存在。
1.6.3体外的异型中和活性
补体非依赖性的体外中和实验按Su等人的方法(38)稍作改进进行。将50μl的用SPG两倍稀释的去补体的单个小鼠血清加到含有105IFU的F血清型NI1品系的50μlSPG溶液中。将该混合液在37℃(5%CO2)下培养30分钟,然后取100μl接种到HBSS(Gibco BRL公司)洗过的Syrian Hamster肾细胞(Hak,ATCC,Rockville,Md)上,37℃(5%CO2)培养2小时。然后,除去培养液,用HBSS液洗细胞,再加入含有10%FCS,50μg/ml庆大霉素和0.5μg/ml环己酮的MEM。37℃下培养24小时之后,固定细胞并用商品化的羊抗rMOMP抗血清(Chemicon公司)和碱性磷酸酶标记的免抗羊抗体(Sigma公司)按上述免疫化学方法检测包涵体。以BCIP/NBT(5-溴-4-氯-3-吲哚磷酸/氮蓝四唑)为反应的酶的底物。IFU是通过用倒置显微镜的5个放大200倍的视野来定量。样品血清每个视野的平均IFU数表示为其相对于以未免疫的小鼠血清为阴性对照而获得的平均IFU数的降低的百分率。中和反应效价(NT50)是以造成传染性降低50%的血清样品的稀释倍数之倒数来计算。
1.7结果
1.7.1重组抗原的表达和鉴定
含有编码成熟的MOMPL2血清型分子的核苷酸序列的DNA片段经PCR扩增,然后插入到正确阅读框并定位于PET15表达载体中。衣原体蛋白核苷酸序列以及和编码5'端His标记肽的多核苷酸序列融合连接如同克隆策略所设计。细胞分级分离之后,表达产物在大肠杆菌的不可溶部分,可见表达产物以不溶性的包含体的形式被表达。将含有沉淀的重组rMOMP用2%SDS缓冲液溶解,而后过金属离子亲和层析柱。镍离子被固定在该柱上,它可以螯合重组rMOMP融合的His标记肽所带有组氨酸残基。SDS-PAGE和Westem blot检测分别证明经过洗涤并在无SDS的缓冲液中洗脱的纯化蛋白的分子量和预期相符,同时能和抗MOMP的单抗和多抗进行免疫反应。透析之后,rMOMP的浓度介于500μg/ml和1mg/ml间而纯度估计达90%。
1.7.2与佐剂一起免疫时rMOMP对小鼠的血清学反应以及异型攻击后生殖力的影响。
用以测评各种带有佐剂的MOMP的预防能力的实验结果见表1。组4和组5是用5μg MPL和10μg QS21作为佐剂同rMOMP一起皮下免疫。在组4中,带有佐剂的重组蛋白在SB62乳化液中制备,其中含有油相鲨烯和α-生育酚,还有吐温80作为表面活性剂。组6也将rMOMP与相同于组4的SB62乳化液一起皮下免疫,但其中不含免疫刺激物。同时设有三个对照组:组1未经任何处理;组2是模拟免疫组,只用免疫刺激物和SB62乳化液一起免疫;组3象组4一样免疫,但这是为了模拟感染。组2,4,5和6用异型的沙眼衣原体品系NI1行免疫攻击。
1.7.3免疫对血清学反应的影响
为了测评各种配方的免疫原性,在第二次接种后4周(攻击日),取血清用ELISA法测IgG效价并计算出每组的平均几何滴度(AMT)。两次5μg的rMOMP注射免疫引起了高水平的rMOMP IgG。如表1,几何平均滴度(AMT)在组4和组6中其实相等,但和免疫刺激因子及乳化液结合后则导致了rMOMP特异的IgG平均效价增加了两倍。仅用佐剂的动物模拟免疫(组2)没有明显的抗衣原体重组抗原的抗体效价。各组中,在免疫攻击前收集的阴道洗液中均检测不到特异的rMOMP分泌型IgA。
如表1所示,在含有免疫刺激物的组(组4和组5)与用SB62乳化rMOMP的组(组6)之间,IgG亚类的分布有显著差异。使用MPL+Q21明显提高了IgG2a的相对水平,却降低了IgG1的相对水平;在非乳化的配方中这种现象表现到极至。
为了证实rMOMP特异的IgG对异型的感染品系是否有交叉反应,将样品血清稀释100倍并分别在甲醇固定的感染细胞上进行酶免实验以检测其识别衣原体包涵体的能力。所有来自免疫组的血清均表现出一定的与用于攻击的衣原体品系NI1反应的IgG。因此,在体外的补体非依赖性试验中分别测定这些血清抵抗该品系的中和活性(用模拟免疫组的血清作为阴性对照)。因为没有任何一组血清能明显降低衣原体的传染性,所以与ELISA和酶免实验的结果比,这些结果是不一致的。
1.7.4异型免疫对攻击后生育力的影响
在用rMOMP最后一次免疫(或模拟免疫)8周后并且宫内感染(或模拟感染)4周之后,雌鼠用雄鼠交配两个月。异源的NI1品系对C3H/HeOuJ小鼠生育力的攻击结果用以下参数计算:每组平均产仔数(N)和平均同窝仔大小(M)。同未处理的小鼠比,衣原体接种的组2(模拟免疫)小鼠生育力明显改变。这个结果证明了此实验动物模型的有效性。经免疫和模拟感染过的小鼠(组3)同未处理组相比表现出降低的生育力参数。这个组是用来作为一个对照评估rMOMP配方的预防能力且考虑到了生育力的非病理性改变。如表1所示,免疫刺激因子共接种组(组4和5)同用SB62乳化rMOMP免疫的组(组6)之间生育力有明显差异。组5表现出最佳结果(10只小鼠有7只产仔)和最大的生育力参数;N值达到对照组3的50%,M值与对照组相当。相反,没加免疫刺激物的组6的生育力参数同感染的对照组2的相当。联合了免疫刺激因子和SB62乳浊液的rMOMP免疫也产生了抗不育的保护作用,但SB62浮浊液的应用好象部分地降低了保护率。如此,应用MPL加上QS21能够部分预防上生殖道衣原体感染,并且该现象的最大效应在非乳化的配方中获得。
1.8结论
我们的结果表明以MPL+QS21为佐剂的rMOMP非肠道免疫能够部分地预防由异源衣原体感染小鼠生殖道所造成的不孕;相反,不加两种免疫刺激因子而仅注射SB62乳化的rMOMP不能产生任何预防作用。另一方面,每一抗原配方在所免疫动物的血清中均引起强烈的而相对同型的MOMP特异的总IgG反应;这些抗体能够和用甲醇固定的异型感染品系的衣原体包涵体进行交叉反应,但不能在体外降低衣原体的传染性。刚好在攻击之前,在阴道分泌物中检测不到rMOMP特异的IgA,这同非肠道抗原给药方法所得结果一致。因此,将总的特异性IgG效价或中和效价同各免疫组的病理学结果对比得不出任何显著的有关这些对比之间的关系。
以上研究结果证实重组MOMP联合免疫刺激物MPL+QS21能够诱发预防沙眼衣原体造成不育的免疫保护作用。2.各种MOMP佐剂配方的第二组实验。
2.1实验材料与方法
衣原体和小鼠品系同实施例1中所述的实验使用的一样。
2.2 MOMP制备和配制
生产和利用构建的PET15-MOMP质粒DNA来生产抗原如实施例1所述。为了获得大量不含内毒素的抗原,抗原纯化方法做了一些修改。简要地说是这样:在依据制造商建议的方法进行纯化步骤之前,将10ml His结合树脂(Novagen公司)用25倍体积的2%SDS/6M尿素的水溶液洗涤。将250ml诱导的裂解物离心,收集沉淀;沉淀分别用4M尿素,2M NaCl和2%Zwittergen(Calbiochem公司产品)洗涤,接着溶于Tris-HCl(PH7.9),0.5%SDS,500mM NaCl,5mM咪唑中。抗原由Tris-HCl(PH7.9),100mM咪唑洗脱下来,对5mM Tris-HCl PH7.4彻底透析,用0.22μm无菌滤膜过滤。然后,用鲎裂解物实验(Coatest,Chromogenix)来分析LPS内容物。
2.3配制
除每剂中的MPL量改为10μg外,抗原按实施例1所述进行配制。有一组使用了同时待审的英国专利申请950832.7,9513107.4(称为DQ)报道的改进的QS21来实验,加入的剂量是10μg/剂。更详细地说,疫苗的配制方案是:加抗原到缓冲液,然后加入100nm大小的MPL颗粒;在一分离管中,QS21同由二油酰磷脂酰胆碱(DOPC)和胆固醇组成的小单层脂质体混合(DOPC∶胆固醇=4∶1W/W),因此QS21和胆固醇之比为1∶5(在此条件下,所有的QS21都掺入脂质体膜中);接着将QS21/SUV混合液(称为DQ)加到抗原/MPL混合液中。在这种配方中,抗原和MPL在脂质体外,而QS21在脂质体内。
2.4小鼠输卵管炎模型的接种,生育力,免疫学和组织学随访。
免疫、实验性感染和取样的方案同前述一致,但阴性对照是由含10μgMPL和10μgQS1的乳浊液模拟免疫;另外增加一组抗原和MPL+DQ联合免疫的小鼠。每组有15只小鼠:其中10只同雄鼠交配8周,而另外5只在免疫攻击两周后处死用于组织病理学和免疫细胞化学分析。用以评估各组生育力的参数如下:F(生产过一次或多次的小鼠数除以总小鼠数),M(新生小鼠数(死亡或者活的)除以总的新生仔数)和N(新生鼠数(死的和活的)除以总的鼠数)。
血清和阴道洗液用ELISA检测rMOMP特异的抗体。血清也用来在体外感染中检测F血清型NI1品系衣原体包涵体识别和异源(NI1)感染中和。所用技术均如前述。
上部生殖道(卵巢、输卵管和子宫角顶)在OCT化合物中包埋(组织-TEK,Miles),速冻,10μm大的冷冻切片装于载波片(Superforst,Menzel-glarter)上。风干切片,丙酮固定5分钟,-70℃贮存。进行组织病理分析时,重新水化切片,然后用苏木精(H)和伊红(E)染色。在进行免疫细胞化学分析染色时,切片用PBS重新水化,用100μl含2μg生物素标记的大鼠抗小鼠CD4或CD8单抗(Serotec公司)的PBS培育60分钟,PBS洗两遍,再在2000倍稀释的HRP-链霉抗生物素蛋白(Zymed公司)中重新培育30分钟。洗涤之后,用液体DAB试剂盒(Zymed公司)显色,用苏木精复标记,在丙烯醇中进行永久封固。
2.5 γ干扰素检测
在第0周和第2周用200μl配方在小鼠尾基部皮下注射,对照组用10μgMPL和10μgQS21联合乳化液模拟免疫。小鼠第4周取血进行血清学分析,然后处死,无菌操作取脾脏,收集细胞并制备单细胞悬液,以用1μg/ml rMOMP重新刺激或者用4/ml ConA(Boer-hinger Mannheim)刺激作为对照。在平底24孔板中以每毫升RPMI 1640106响应细胞加上10%胎牛血清(Gibco-BRL公司产品)进行培养。重刺激96小时后收集上清,用商品化的ELISA试剂盒(Cytoscreen,Biosource)检测γ干扰素(IFN)。
2.6结果
2.6.1抗原
透析之后,rMOMP浓度估计约为2mg/ml,污染的内毒素水平低于0.05EU/μgrMOMP。
2.6.2带佐剂的rMOMP免疫对小鼠体液免疫反应,异型攻击后对生育力,以及感染导致的发炎感染的影响。
用于评估不同佐剂辅助下rMOMP的预防能力的实验结果如表2。
2.6.3免疫对体液反应的影响。攻击的当天收集血清和阴道洗液来检测接种后的体液反应。所有的抗原配方产生相近的几何平均滴度(GMT)大约为30,000的抗rMOMP IgG。只用佐剂的模拟免疫动物没有产生明显的抗衣原体重组抗原的抗体效价。任何一组实验动物中,在刚好在攻击前收集的阴道洗液中均检测不到rMOMP特异的分泌型IgA。同型的rMOMP特异的IgG反应揭示出同只用乳浊液中rMOMP激起的反应相比,MPL+QS21或DQ的存在提高了IgG2a的相对水平。所有的免疫后血清都含有可同甲醇固定的血清种FC沙眼衣原体品系NI1(即用以免疫攻击)的包涵体反应的IgG。
2.6.4攻击后的异型免疫对生育力的影响。用异源的NI1品系对小鼠生育的攻击结果用实验步骤中定义的参数F、N和M来定量。计算出的各组在交配期的这些参数值见表1。和模拟感染组相反,对模拟免疫组的感染几乎导致完全不育。这就说明观察到的不育是由于沙眼衣原体所致,而不是由于对动物的操作。两种佐剂都加的rMOMP免疫组,即MPL+DQ配方的抗原产生了部分的保护作用:F和N多数的值大约达到了模拟感染组(阳性对照)最高值的80%。相反,在攻击前用乳浊液中配置的rMOMP免疫所得的生育力参数F和N值较低。
2.6.5攻击后的组织病理学变化
在生育力实验的组织病理学副实验中,组织切片经传统HE着色发现在模拟免疫组和接种组间,输卵管和卵巢的炎症伤痕处无明显的差异。可是用免疫细胞化学染色观察T细胞亚群频率时,CD4阳性T细胞只在MPL+DQ接种组中发现(4/4小鼠),而CD8阳性T细胞在免疫和模拟免疫组均有(表3)。因此在这实验中,只在MPL+DQ接种组中发现了CD4阳性的浸润T细胞并且在生育力研究的副实验中,这也是唯一有保护性的组。
2.6.6该配方在体外再刺激时,对γ干扰素分泌的影响。
抗原配方诱导的细胞活性用单独的实验检测(见表4)。一方面,将rMOMP联合MPL+QS21或MPL+DQ接种的动物的脾细胞分离出来并用抗原在体外重刺激后,发现其培养上清中就有浓度和在同期用4μg/ml的刀豆球蛋白A刺激后的相当的γ-干扰素。另一方面,从模拟免疫或接种了无免疫刺激因子的rMOMP的动物中分离的细胞却不能产生可测到的γ-干扰素,而其用ConA共培养的副实验总表现出此细胞因子阳性。对从各组中收集的血清样进行血清学分析揭示出γ干扰素的分泌同抗原特异的IgG2a比率增高相关。
结论
总之,攻击试验和γ干扰素检测的实验的数据表明重组MOMP同两种佐剂MPL和QS21或IDQ的结合物在小鼠体内联合免疫能导致抗原特异的Th1样免疫反应(这由γ干扰素分泌和IgG2a比例的升高的来检测),并因此能预防沙眼衣原体引起的不育症。
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表1监控12周的免疫组和对照组小鼠的血清学和生育力检测
组号免疫/感染方案 | rMOMP特异的总IgG的平均效价 | IgG1∶IgG2a∶gG2b的平均比率 | F血清型包涵体检测a | 中和反应(NT 50)b | 产仔鼠数:组内鼠数 | N(组内新生鼠数:组内鼠数) | M(平均窝仔大小) |
Gl未处理 | <100 | ND | No | <20 | 8/8 | 10.9 | 4.7 |
G2MPL+QS21+SB62感染 | <100 | ND | No | <20 | 3/10 | 1.3 | 2.3 |
G3MOMP+MPL+QS21+SB62模拟感染 | 34800 | 44.6∶42.6∶12.8 | γes | <20 | 7/8 | 8.7 | 3.7 |
G4MOMP+MPL+QS21+SB62感染 | 43000 | 58.3∶35∶12.7 | γes | <20 | 7/10 | 3.4 | 2.8 |
G5MOMP+MPL+QS21感染 | 113000 | 28.9∶66.6∶4.5 | γes | <20 | 7/10 | 4.5 | 3.8 |
G6MOMP+SB62感染 | 43000 | 83∶14.6∶2.4 | γes | <20 | 2/10 | 0.6 | 3 |
表2
组别免疫/感染方案 | rMOMP-特异的IgGGMT | 同型比率IgG2a;IgG1;IgG2b | F:可育鼠比例(%) | N:每只小鼠平均产仔数 | M:平均仔大小 |
G1(阴性对照)-(SC)MPL+QS21+SB62/感染 | <100 | ND | 1/8(12.5) | 0.8 | 4 |
G2(阳性对照)rMOMP L2(sc)MPL+QS21+SB62/模拟感染 | 23900 | 44;52;4 | 9/10(90) | 6.2 | 3.6 |
G3rMOMP L2(sc)MPL+QS21/感染 | 32820 | 49;47;4 | 4/9(44) | 1.7 | 3 |
G4rMOMP L2(sc)SB62/感染 | 41415 | 14;83;3 | 3/10(30) | 1.9 | 4.75 |
G5rMOMP L2(sc)DQ+MPL/感染 | 30830 | 32;65;3 | 6/8(75) | 4.8 | 3.3 |
sc:皮下注射
GMT:几何平均效价
MD:未做
表3
组别免疫/感染方案 | 鼠号 | CD8阳性T细胞得分 | CD4阳性T细胞得分 | ||
输卵管 | 卵巢 | 输卵管 | 卵巢 | ||
G1(阴性对照)-(SC)MPL+QS21+SB62/感染 | 1234 | +/+++/++NDND | +++/++NDND | --NDND | --NDND |
G2(阳性对照)rMOMP L2(sc)MPL+QS21+SB62/模拟感染 | 1234 | ---- | ---- | ---- | ---- |
G3rMOMP L2(sc)MPL+QS21/感染 | 1234 | ++/+++/+++/- | +++/-- | ---- | ---- |
G4rMOMP L2(sc)SB62/感染 | 1234 | +/-+/+++/+++/++ | +/-+++ | ND--- | ND--- |
G5rMOMP L2(sc)DQ+MPL/感染 | 1234 | +/+++++/++++ | ++++++ | +/---+/- | +/+++/-++/++ |
sc:皮下途径
ND:未做
CD4或CD8阳性T细胞点数是在所研究细胞类型中从没有细胞浸润(一)到最大数量细胞
浸润(+++)而分级评定的
表4
组别免疫方案 | rMOMP-特异的IgG GMT | 同型比率IgG2a;IgG2b;IgG1 | ConA(4μg/ml)再刺激后的γ-干扰素(pg/ml) | rMOMP(4μg/ml)再刺激后的γ-干扰素(pg/ml) |
G1(阴性对照)-(SC)MPL+QS21+SB62 | ND | ND | 555 | <25 |
G2rMOMP L2(sc)SB62 | 18000 | 8.8;3.5;87.7 | 353 | <25 |
G3rMOMP L2(sc)MPL+QS21 | 49000 | 70;6.8;23.2 | 397 | 503 |
G4rMOMP L2(sc)DQ+MPL | 42000 | 57.5;6;36.5 | 461 | 258 |
sc:皮下途径
GMT:几何平均效价
ND:未做
Claims (6)
1.一种包括来源于衣原体的MOMP蛋白并带有QS21和3DMPL的疫苗组合物。
2.按照权利要求1所述的疫苗,其中的蛋白来源于L2血清型。
3.按照权利要求1或者2所述的疫苗,其中还含有一种来源于不同血清型的蛋白。
4.按照权利要求1所述的疫苗,其中还含有固醇。
5.按照权利要求4所述的疫苗,其中的固醇是胆固醇。
6.按照权利要求5所述的疫苗,其中的QS21和含有胆固醇的脂质体相联。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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GB9506863.1 | 1995-04-03 | ||
GBGB9506863.1A GB9506863D0 (en) | 1995-04-03 | 1995-04-03 | Vaccines |
Publications (1)
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CN1185740A true CN1185740A (zh) | 1998-06-24 |
Family
ID=10772426
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Application Number | Title | Priority Date | Filing Date |
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CN96194300A Pending CN1185740A (zh) | 1995-04-03 | 1996-04-01 | 衣原体疫苗 |
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EP (1) | EP0820304B1 (zh) |
JP (2) | JPH11503142A (zh) |
KR (1) | KR19980703666A (zh) |
CN (1) | CN1185740A (zh) |
AT (1) | ATE193974T1 (zh) |
AU (1) | AU700548B2 (zh) |
BR (1) | BR9604853A (zh) |
CA (1) | CA2215520C (zh) |
CZ (1) | CZ313497A3 (zh) |
DE (1) | DE69608959T2 (zh) |
DK (1) | DK0820304T3 (zh) |
ES (1) | ES2149466T3 (zh) |
GB (1) | GB9506863D0 (zh) |
GR (1) | GR3033764T3 (zh) |
HK (1) | HK1008495A1 (zh) |
HU (1) | HUP9801572A3 (zh) |
NO (1) | NO974561L (zh) |
NZ (1) | NZ306443A (zh) |
PL (1) | PL322620A1 (zh) |
PT (1) | PT820304E (zh) |
TR (1) | TR199701113T1 (zh) |
WO (1) | WO1996031236A1 (zh) |
ZA (1) | ZA962616B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100467600C (zh) * | 2000-07-20 | 2009-03-11 | 科里克萨有限公司 | 用于治疗和诊断衣原体感染的化合物和方法 |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9626864D0 (en) * | 1996-12-24 | 1997-02-12 | Smithkline Beecham Biolog | Vaccine |
US7459524B1 (en) | 1997-10-02 | 2008-12-02 | Emergent Product Development Gaithersburg Inc. | Chlamydia protein, sequence and uses thereof |
US6306404B1 (en) | 1998-07-14 | 2001-10-23 | American Cyanamid Company | Adjuvant and vaccine compositions containing monophosphoryl lipid A |
GB9819898D0 (en) * | 1998-09-11 | 1998-11-04 | Smithkline Beecham Plc | New vaccine and method of use |
US6635261B2 (en) | 1999-07-13 | 2003-10-21 | Wyeth Holdings Corporation | Adjuvant and vaccine compositions containing monophosphoryl lipid A |
US20010048927A1 (en) | 2000-02-01 | 2001-12-06 | Richard Stephens | Porin B (PorB) as a therapeutic target for prevention and treatment of infection by chlamydia |
US7105171B2 (en) | 2002-03-07 | 2006-09-12 | The Regents Of The University Of California | Porin B (PorB) as a therapeutic target for prevention and treatment of infection by Chlamydia |
US8541007B2 (en) | 2005-03-31 | 2013-09-24 | Glaxosmithkline Biologicals S.A. | Vaccines against chlamydial infection |
MX2007012108A (es) * | 2005-03-31 | 2007-12-05 | Glaxosmithkline Biolog Sa | Vacunas contra infeccion por chlamydia. |
CN103068837B (zh) | 2010-05-28 | 2015-06-24 | 斯匹遐生物技术公司 | 嵌合momp抗原、方法和使用 |
CN113940994B (zh) * | 2021-11-09 | 2023-09-15 | 南华大学 | 壳聚糖-Pickering乳液白细胞介素12佐剂体系的制备方法及其应用 |
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NL9000207A (zh) * | 1990-01-29 | 1991-08-16 | Duphar Int Res | |
WO1992010203A1 (en) * | 1990-12-10 | 1992-06-25 | Alving Carl R | A vaccine against cholesterol to prevent hypercholesterolemia and atherosclerosis |
CH682455A5 (fr) * | 1991-07-25 | 1993-09-30 | Serge Liotet | Composition vaccinale. |
JPH05339169A (ja) * | 1992-03-03 | 1993-12-21 | Dai Ichi Seiyaku Co Ltd | 経口ワクチン |
PT761231E (pt) * | 1992-06-25 | 2000-06-30 | Smithkline Beecham Biolog | Composicao de vacina contendo adjuvantes |
-
1995
- 1995-04-03 GB GBGB9506863.1A patent/GB9506863D0/en active Pending
-
1996
- 1996-04-01 TR TR97/01113T patent/TR199701113T1/xx unknown
- 1996-04-01 JP JP8529985A patent/JPH11503142A/ja not_active Withdrawn
- 1996-04-01 AT AT96912001T patent/ATE193974T1/de not_active IP Right Cessation
- 1996-04-01 CZ CZ973134A patent/CZ313497A3/cs unknown
- 1996-04-01 DK DK96912001T patent/DK0820304T3/da active
- 1996-04-01 PT PT96912001T patent/PT820304E/pt unknown
- 1996-04-01 BR BR9604853A patent/BR9604853A/pt not_active Application Discontinuation
- 1996-04-01 HU HU9801572A patent/HUP9801572A3/hu unknown
- 1996-04-01 ES ES96912001T patent/ES2149466T3/es not_active Expired - Lifetime
- 1996-04-01 KR KR1019970707065A patent/KR19980703666A/ko not_active Application Discontinuation
- 1996-04-01 CN CN96194300A patent/CN1185740A/zh active Pending
- 1996-04-01 NZ NZ306443A patent/NZ306443A/xx unknown
- 1996-04-01 AU AU54999/96A patent/AU700548B2/en not_active Ceased
- 1996-04-01 PL PL96322620A patent/PL322620A1/xx unknown
- 1996-04-01 CA CA002215520A patent/CA2215520C/en not_active Expired - Fee Related
- 1996-04-01 DE DE69608959T patent/DE69608959T2/de not_active Expired - Lifetime
- 1996-04-01 EP EP96912001A patent/EP0820304B1/en not_active Expired - Lifetime
- 1996-04-01 WO PCT/EP1996/001463 patent/WO1996031236A1/en not_active Application Discontinuation
- 1996-04-02 ZA ZA962616A patent/ZA962616B/xx unknown
-
1997
- 1997-10-02 NO NO974561A patent/NO974561L/no unknown
-
1998
- 1998-07-28 HK HK98109493A patent/HK1008495A1/xx not_active IP Right Cessation
-
2000
- 2000-06-23 GR GR20000401460T patent/GR3033764T3/el unknown
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2007
- 2007-07-12 JP JP2007183164A patent/JP2007308508A/ja not_active Ceased
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN100467600C (zh) * | 2000-07-20 | 2009-03-11 | 科里克萨有限公司 | 用于治疗和诊断衣原体感染的化合物和方法 |
Also Published As
Publication number | Publication date |
---|---|
ZA962616B (en) | 1996-11-27 |
DE69608959D1 (de) | 2000-07-27 |
HK1008495A1 (en) | 1999-05-14 |
BR9604853A (pt) | 1998-06-16 |
CA2215520C (en) | 2008-01-22 |
JP2007308508A (ja) | 2007-11-29 |
GB9506863D0 (en) | 1995-05-24 |
EP0820304A1 (en) | 1998-01-28 |
GR3033764T3 (en) | 2000-10-31 |
CA2215520A1 (en) | 1996-10-10 |
DK0820304T3 (da) | 2000-08-28 |
MX9707638A (es) | 1998-06-28 |
PL322620A1 (en) | 1998-02-02 |
HUP9801572A2 (hu) | 1998-10-28 |
ES2149466T3 (es) | 2000-11-01 |
PT820304E (pt) | 2000-11-30 |
NZ306443A (en) | 1999-02-25 |
CZ313497A3 (cs) | 1998-03-18 |
ATE193974T1 (de) | 2000-07-15 |
AU5499996A (en) | 1996-10-23 |
NO974561D0 (no) | 1997-10-02 |
TR199701113T1 (xx) | 1998-03-21 |
NO974561L (no) | 1997-11-26 |
EP0820304B1 (en) | 2000-06-21 |
WO1996031236A1 (en) | 1996-10-10 |
AU700548B2 (en) | 1999-01-07 |
JPH11503142A (ja) | 1999-03-23 |
KR19980703666A (ko) | 1998-12-05 |
HUP9801572A3 (en) | 1999-04-28 |
DE69608959T2 (de) | 2000-11-16 |
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