CN102741414A - 通过重组酵母口服/粘膜接种疫苗的方法 - Google Patents
通过重组酵母口服/粘膜接种疫苗的方法 Download PDFInfo
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Abstract
本发明涉及重组酵母细胞的产生，所述重组酵母细胞被用于接种以及疫苗特别是通过服食而口服接种疫苗。
Description
对传染病的防范在人类医学以及兽医学中依然是巨大的挑战。对于病毒感染而言情况尤其复杂，与细菌感染相反，其通常不能用广谱的有效物质来控制且会引起巨大的经济损失。针对病毒性疾病的新的有效接种疫苗策略的开发因而具有突出的重要意义。
传统上针对病毒性疾病的预防性以及治疗性接种疫苗是经“减毒的”，即利用通过诱变改造的具有显著降低或者缺失的毒力的病毒(“活疫苗”)、或灭活的病毒(“死疫苗”)。近来，也建立了日益增加的所谓“亚单位疫苗”或者“标记疫苗”，其中使用给定的经遗传工程改造的病原体“主要抗原(Hauptantigen)”。术语“标记疫苗”意指通过随后的诊断分析可清楚地将经接种疫苗的个体与自然感染的个体区别开。对于主要抗原可理解为例如病毒包膜或病毒衣壳的蛋白，其在缺乏完整的病毒颗粒时可在宿主中诱导体液和/或细胞免疫应答，作为其结果病毒感染可被阻断或者被控制。“亚单位接种疫苗”需要先将主要抗原进行表征。这些蛋白的生产(表达)及免疫原性配制对于接种疫苗的过程发挥着关键的作用，特别是病毒的外壳蛋白大多数不溶于水并且在细菌表达系统中仅有很少的能被重组表达。以纯化的形式获得“亚单位”疫苗并确保其持久性的方法相应地成本较高。
酵母作为用于重组蛋白的表达系统将细菌系统经济的优点与高等细胞典型的组织形式结合起来。它们通过细胞内膜系统来区室化(kompartimentiert)，这从根本上将其与细菌宿主系统区别开并且使得膜锚定的病毒外壳蛋白或者甚至是整个病毒衣壳能够表达。此外，构成酵母细胞壁的β-葡聚糖、葡萄糖聚合物具有长久以来已知的免疫刺激效应。由此，任务是开发一种方法，其中完整的酵母可被用于接种疫苗。此问题由如下方式解决，通过基因工程的方法将免疫原决定簇(immunogene Determinanten)的基因引入非病原酵母的基因组并表达，并将此重组酵母细胞直接用于接种疫苗以及特别是通过服食而口服接种疫苗。
乳酸克鲁维氏酵母(Kluyveromyces lactis)
乳酸克鲁维氏酵母属于具有GRAS(通常认为安全)级别的所谓的食品级酵母。与作为食品添加剂经过了上千年测试和验证的面包酵母一样，在奶制品中通常的代表性酵母乳酸克鲁维氏酵母对于食品工业也是安全无害的。
口服/粘膜接种疫苗
对于疫苗/抗原的施用有如下的方式可选择：皮下、肌内、肠胃外、或粘膜/口服。而在前三种方式中抗原直接进入血液或淋巴管道，对于粘膜/口服应用抗原的暴露则是通过支气管或胃肠道粘液膜(粘膜)进行的。因而术语粘膜/口服包括抗体的鼻/支气管施用以及口服施用。支气管和肠粘膜均持久地暴露于病原体并且其本身是感染的主要接受屏障。与粘膜表面特别是粘膜肠上皮结合的免疫系统包含了例如人体内所有免疫活性细胞的约90％。在肠粘膜中所谓的诱导位点，抗原的接受和呈递通过树突细胞和“淋巴集结”的M-细胞(总体上称为MALT，粘膜相关性淋巴样组织)而进行，但根据近来的发现也通过肠细胞和肠上皮细胞进行。在鼻/支气管粘膜上存在类似的组合(BALT，支气管相关性淋巴样组织)。在免疫应答缓和(Abklingen)后，在整个生物体以及在粘膜特异性记忆免疫细胞的所谓效应位点通常产生针对原始抗原/病原体介导的长效防护。
口服/粘膜接种疫苗方法的优势。与肠胃外免疫相比，口服/粘膜免疫需要使用显著较高的量的抗原。与肠胃外接种介苗相反，通过口服/粘膜接种疫苗可以在诱导全身免疫应答之外还在粘膜的效应位点诱导局部免疫应答。特别是对于(也)通过粘液膜传播的病原体(如牛病毒性腹泻病毒BVDV和猪瘟病毒CSFV，见下文)而言，粘膜/口服接种疫苗具有产生有活性且长效免疫的潜力。口服/粘膜接种疫苗的重要优势还有良好的接受性和经济性。在最理想的情况下所述疫苗可以低成本生产并方便地与食物一起施用。
用表达病毒抗原的酵母菌株进行的粘膜/口服接种疫苗不仅仅安全无害，还能够具有额外的健康增强和辅佐效应。
现在的目标是开发出基于乳酸克鲁维氏酵母的表达系统，其允许外源基因定向整合到酵母基因组中并因此使得能够形成适当的抗原。进一步的目标是表达特定病毒抗原的重组乳酸克鲁维氏酵母菌株，其用于接种疫苗以及特别是用于粘膜/口服接种疫苗。
根据本发明，通过遗传工程的方法产生乳酸克鲁维氏酵母的菌株(优选VAK367-D4及其变体)，其允许外源基因定向整合至酵母基因组的LAC4基因座而不必引入额外的DNA序列(选择性标记或其它的)。该重组酵母菌株稳定无选择压力并且在发酵的条件下能够被培养至高密度。外源基因的表达可以通过乳糖或半乳糖的剂量来诱导，或者在关闭调节基因KIGAL80后组成性地激活。外源基因的表达可间接地通过内源报道基因的表达而进行定量。
产生了在乳酸克鲁维氏酵母菌株VAK367-D4上构建的一系列变体。通常这些变体可诱导地表达大量的蛋白、或该蛋白的结构域、或与不同种(artfremd)蛋白结构域融合的该蛋白的结构域。所关联的不同种蛋白结构域用于进行免疫应答的定向刺激(辅佐目的)或者用于所表达的外源蛋白在酵母细胞内的定向区室化(Kompartimentierung)。在辅佐效应之外，所表达的外源蛋白的区室化对于表达的优化或者表达产物的形成很重要。这些重组乳酸克鲁维氏酵母菌株之一(参见实施方案)已成功地被用于粘膜/口服接种疫苗。
实施方案：
1.乳酸克鲁维氏酵母菌株VAK367-D4(Met-ura3 lac4::ScURA3)的产生。
用于异源表达外源蛋白的起始菌株VAK367具有如下特性：其允许培养物达到高细胞密度而不伴有细胞内蛋白可检测的释放。在这点上此菌株与许多紧密关联的乳酸克鲁维氏酵母菌株不同。菌株VAK367通过两轮的诱变而衍生自菌株CBS 2359(Centraalbureau voor Schimmelcultureshttp://www.fungalbiodiversitycentre.com)，并且是氨基酸甲硫氨酸和核碱基尿嘧啶的营养缺陷型。可通过遗传工程的方法从菌株VAK367衍生出菌株VAK367-D4(保藏于Braunschweig的Deutschen Sammlung vonMikroorganismen und Zellkulturen GmbH(DSMZ))，其中借助质粒pD4-2经ScURA3基因而取代LAC4基因从+358至+1181的序列。该菌株VAK367-D4允许外源基因整合至LAC4基因座而无附加的标记，其要在乳糖上生长来筛选。这需要使用适当的整合载体如Klp3(见下文)，通过同源重组取代分裂盒(Disruptionskassette)，从而在失去ScURA3标记时重组完整的LAC4基因(图1)。
2.允许外源蛋白可诱导地表达的整合载体的产生。
载体Klp3
载体Klp3是一种基于YRp7的大肠杆菌(E.coli)载体，由于ARS1序列被缺失，其在酵母中不能自主复制。Klp3含有使LAC4基因座能够通过同源重组进行整合的乳酸克鲁维氏酵母序列(LAC4的上游区域和LAC4阅读框的5’端)。在LAC4启动子和转录起点之间插入一个DNA区段，其含有TEF1终止子和KIGAL80启动子。由此LAC4阅读框受到KIGAL80启动子的控制，该启动子通过转录因子KIGal4与LAC4启动子共调节(Zenke et al.1993，Molecular and Cellular Biology，13：7566-7576)。此构造使得可以通过测量LAC4编码的β-半乳糖苷酶来追踪外源蛋白的表达。Klp3允许将外源基因整合至LAC4启动子和TEF1终止子之间独特的Sal1界面。为整合表达盒，用Hpa1或EcoR1消化Klp3并将其转化至克鲁维氏酵母VAK367-D4。此处来自大肠杆菌载体部分的表达盒被分隔，从而使所得的菌株不含有细菌序列。
质粒Klp3-E2-1(9437bp)(图2)
将病毒BVDV的编码病毒结构蛋白E2的基因区段作为Sal1-Xho1片段插入到LAC4启动子和TEF1终止子之间的Sal1界面。下游为KIGAL80启动子，其与LAC4 ORF的5’端融合。
将该质粒用Hpa1剪切并将较大的Hpa1片段与E2-ORF经同源重组而染色体整合。由此基因座lac4::URA3被取代并且完整的LAC4基因被重组。筛选通过在乳糖培养基上生长来实现。URA3基因的丢失通过尿嘧啶的营养缺陷来确认。所述基因座的序列通过DNA-测序来确认。(序列表1)
3.来自BVDV和CSFV的E2主要抗原的形成。
所表征的主要抗原来自BVDV(牛病毒性腹泻病毒)——牛病毒性腹泻和粘膜疾病(BVD/MD)的病原体，以及来自CSFV(猪瘟病毒)——猪瘟(CSF)的病原体。此处是“包膜”(病毒外壳整合的)蛋白E2。在缺乏病毒颗粒时E2也诱导大量的体液免疫应答，也即有效的病毒中和抗体的形成。通过遗传工程形成E2可进一步加强该蛋白的免疫原性潜力，并且也产生细胞免疫应答。针对单一的、或者经遗传工程形成的E2蛋白结构域的特异的且专有的免疫应答，使得可以将经接种疫苗的动物与野外病毒感染的动物区别开，例如通过ELISA的方法。
4.构建表达BVDV E2蛋白的乳酸克鲁维氏酵母菌株。
通过本发明的技术，产生了一种乳酸克鲁维氏酵母菌株VAK367-E2-1。在该菌株中BVDV基因组(菌株CP7)的区段被整合入酵母基因组。所述的BVDV基因区段含有BVDV基因组的包含E2蛋白的区域以及相邻的E1和p7编码区的部分。所述E1和p7区含有用于E2蛋白(序列表2)的正确加工所必须的信号序列。BVDV E2蛋白的正确加工(成熟)经信号酶而实现。
通过特别开发的免疫荧光探测方法，可以确定E2在乳酸克鲁维氏酵母菌株VAK367-E2-1的细胞中的表达。经开发的特别用于BVDV E2的检测的ELISA-方法使得可以对异源表达的抗原进行探测和定量。类似的ELISA-方法可用于对经免疫的动物中抗体的效价进行准确定量。病毒中和的方法以及对抗体和T-细胞进行表征的方法也被用作常规方法。
一种新颖的qRT-PCR方法使得可以对来自血清和细胞培养物上清的BVDV RNA基因组进行检测和定量。
5.对乳酸克鲁维氏酵母菌株VAK367-E2-1在粘膜/口服免疫研究中有效性的检测。
研究1
在动物实验中，于标准化的条件下将首次用于实验的乳酸克鲁维氏酵母乳液应用于大量的实验小鼠。在此使用不同的免疫方案。主要的标准是所提供的酵母的不同的量(每天所摄取食物的3％至最多8％的比例)和不同的“加强免疫间隔”。
结果：
(i)证明了对乳酸克鲁维氏酵母通常的耐受性：口服给予酵母乳液未对动物的状况引起可观测的改变。
(ii)乳酸克鲁维氏酵母的口服给予导致了针对某些酵母蛋白的可清楚地探测的体液免疫应答。提供Western-血液-方法发现用克鲁维氏酵母喂养的动物与对照动物相比具有显著的且特异性的针对酵母蛋白的抗体应答。因而酵母蛋白本身具有免疫原性效应。在口服应用乳酸克鲁维氏酵母可以产生免疫应答的原则性证明之外，还表明通过口服给予组合了重组抗原的酵母可以实现额外的辅佐效应。
研究2
在进一步的动物实验中，用优化的免疫方案(见研究1)并用重组乳酸克鲁维氏酵母VAK367-E2-1菌株对大量的小鼠进行口服接种疫苗。
结果：
(i)通过特定的ELISA-方法，探测到经乳酸克鲁维氏酵母VAK367-E2-1菌株接种疫苗的小鼠与对照小鼠(用首次用于实验的乳酸克鲁维氏酵母接种疫苗)相比有抗BVDV E2抗体的形成。
(ii)在牛培养细胞中BVDV的中和测试中，还是与来自对照小鼠的血清相比，来自经乳酸克鲁维氏酵母VAK367-E2-1菌株免疫的小鼠的抗血清可被发现具有中和效应。
(iii)通过使用佐剂如CpG-ODN和QuilA可以实现免疫应答的增强。
(iv)通过用重组乳酸克鲁维氏酵母VAK367-E2-1菌株进行粘膜/口服免疫，可以实现有效的防护。这可以在用表达BVDV E2蛋白的重组接种疫苗的攻击实验中得到证明。
研究的结果表明，根据本发明，通过产生乳酸克鲁维氏酵母菌株及该菌株的变体，可以将没有另外的选择性标记的外源基因定向整合进其中。所得到的该乳酸克鲁维氏酵母菌株的衍生物可以表达的目的被用于外源蛋白随后的生化合成。所述重组乳酸克鲁维氏酵母菌株的细胞可直接被用于粘膜/口服或者肠胃外免疫。
Claims (28)
1.通过重组酵母口服/粘膜接种疫苗的方法，特征在于通过使用遗传工程的方法产生一种酵母菌株，其使外源基因能够定向整合以及使外源蛋白能够表达。
2.权利要求1的方法，特征在于能够产生酵母菌株的重组变体。
3.权利要求1和2的方法，特征在于产生乳酸克鲁维氏(Kluyveromyceslactis)酵母的重组菌株。
4.权利要求1-3的方法，特征在于产生乳酸克鲁维氏酵母的重组菌株并用作表达系统。
5.权利要求1-4的方法，特征在于产生乳酸克鲁维氏酵母的重组变体，其允许可诱导的外源基因表达。
6.乳酸克鲁维氏酵母的菌株VAK367-D4及该菌株的变体。
7.一或多种上述权利要求中的方法，特征在于所述外源基因的整合在没有额外的载体序列或选择性标记的情况下进行。
8.一或多种上述权利要求中的方法，特征在于所述外源基因表达是组成性的。
9.一或多种上述权利要求中的方法，特征在于可通过内源报道基因的表达间接地对所述外源基因表达进行定量。
10.一或多种上述权利要求中的方法，特征在于所述外源基因使具有抗原特性的蛋白能够表达。
11.一或多种上述权利要求中的方法，特征在于所述外源基因使具有抗原特性的病毒蛋白能够表达。
12.一或多种上述权利要求中的方法，特征在于所述外源基因使病毒的结构蛋白能够表达。
13.一或多种上述权利要求中的方法，特征在于所述外源基因使黄病毒科(Flaviviridae)、双核糖核酸病毒科(Birnaviridae)和正粘病毒科(Orthomyxoviridae)的病毒结构蛋白能够表达。
14.一或多种上述权利要求中的方法，特征在于所述外源基因使牛病毒性腹泻病毒(BVDV)的结构蛋白能够表达。
15.一或多种上述权利要求中的方法，特征在于权利要求1至6的重组酵母菌株变体可诱导地或组成性地表达大量的蛋白或该蛋白的结构域或该蛋白与不同种蛋白结构域融合的结构域。
16.一或多种上述权利要求中的方法，特征在于使外源蛋白能生化合成。
17.根据一或多种上述权利要求中方法生产疫苗的方法，特征在于其在低温运输系统外具有长保存期。
18.一或多种上述权利要求中的方法，特征在于通过施用权利要求1至6的一种重组菌株的完整酵母细胞，产生针对表达的外源蛋白的特异性免疫。
19.权利要求17的方法，特征在于通过吸入、支气管、口服、鼻部施用权利要求1至6的一种重组菌株的完整酵母细胞，而产生针对表达的外源蛋白的特异性免疫。
20.权利要求17的方法，特征在于，权利要求1至6中的一种重组菌株的完整酵母细胞的施用，也可以用其它方式进行。
21.权利要求17-19的方法，特征在于生化合成的外源蛋白使得能够产生特异性免疫。
22.特异性的ELISA方法，用于对针对上述权利要求之一中蛋白的免疫应答进行探测和定量。
23.根据权利要求17的ELISA方法，用于对针对BVDV E2蛋白的免疫应答进行探测和定量。
24.用于检测来自个体血清的中和抗体的方法，其中所述个体由上述权利要求之一中的方法免疫。
25.用于检测针对来自个体血清的BVDV E2蛋白的中和抗体的方法，其中所述个体由上述权利要求之一中的方法免疫。
26.在用抗原攻击后对上述权利要求之一中的免疫进行检测的方法。
27.在用病毒抗原攻击后对上述权利要求之一中的免疫进行检测的方法。
28.在用BVDV E2攻击后对上述权利要求之一中的免疫进行检测的方法
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| DE102008057451A DE102008057451A1 (de) | 2008-11-14 | 2008-11-14 | Verfahren zur oralen Vakzinierung mittels rekombinanter Hefen |
| DE102008057451.1 | 2008-11-14 | ||
| PCT/DE2009/001623 WO2010054649A2 (de) | 2008-11-14 | 2009-11-13 | Verfahren zur oralen/mukosalen vakzinierung mittels rekombinanter hefen |
Publications (2)
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| CN102741414A true CN102741414A (zh) | 2012-10-17 |
| CN102741414B CN102741414B (zh) | 2015-11-25 |
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| CN200980150388.0A Active CN102741414B (zh) | 2008-11-14 | 2009-11-13 | 通过重组酵母口服/粘膜接种疫苗的方法 |
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| US (1) | US9885052B2 (zh) |
| EP (1) | EP2403947B1 (zh) |
| JP (1) | JP5930716B2 (zh) |
| CN (1) | CN102741414B (zh) |
| DE (2) | DE102008057451A1 (zh) |
| DK (1) | DK2403947T3 (zh) |
| ES (1) | ES2669018T3 (zh) |
| PL (1) | PL2403947T3 (zh) |
| WO (1) | WO2010054649A2 (zh) |
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| DE102011121069A1 (de) * | 2011-12-13 | 2013-06-13 | Martin-Luther-Universität Halle-Wittenberg | Vakzinierung mittels rekombinanter Hefe durch Erzeugung einer protektiven humoralen Immunantwort gegen definierte Antigene |
| KR101876535B1 (ko) * | 2012-06-14 | 2018-07-09 | 베트올 (주) | 소바이러스성 설사병 바이러스의 탐지용 항체, 이를 이용한 항원 검출 방법, 및 이를 포함하는 탐지키트 |
| DE102017012109A1 (de) | 2017-12-27 | 2019-06-27 | Martin-Luther-Universität Halle-Wittenberg | Optimiertes Wirts-/Vektorsystem zur Erzeugung protektiver mono- und multivalenter subunit-Vakzine auf Basis der Hefe Kluyveromyces lactis |
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| FR2679920A1 (fr) * | 1991-08-02 | 1993-02-05 | Rhone Poulenc Rorer Sa | Levures recombinantes hautement stables pour la production de proteines recombinantes, leur preparation et leur utilisation. |
| US5830463A (en) * | 1993-07-07 | 1998-11-03 | University Technology Corporation | Yeast-based delivery vehicles |
| US5541102A (en) * | 1994-09-02 | 1996-07-30 | Board Of Regents, University Of Nebraska-Lincoln | Bovine cell line resistant to in vitro infection by bovine viral diarrhea virus and all other known pestiviruses |
| US6174667B1 (en) * | 1997-09-23 | 2001-01-16 | Cornell Research Foundation, Inc. | Bovine viral diarrhea virus serum antigen capture |
| EP1174506A1 (en) * | 2000-06-28 | 2002-01-23 | Stichting Dienst Landbouwkundig Onderzoek | C-terminal Erns peptide and analogues thereof |
| AU2002225681A1 (en) * | 2000-11-15 | 2002-05-27 | Globe Immune, Inc. | Yeast-dentritic cell vaccines and uses thereof |
| JP4840774B2 (ja) * | 2004-09-10 | 2011-12-21 | 旭硝子株式会社 | 経口投与ワクチン |
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2008
- 2008-11-14 DE DE102008057451A patent/DE102008057451A1/de not_active Withdrawn
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- 2009-11-13 JP JP2011535875A patent/JP5930716B2/ja active Active
- 2009-11-13 DE DE112009003267T patent/DE112009003267A5/de not_active Withdrawn
- 2009-11-13 CN CN200980150388.0A patent/CN102741414B/zh active Active
- 2009-11-13 US US13/129,267 patent/US9885052B2/en active Active
- 2009-11-13 EP EP09808976.6A patent/EP2403947B1/de not_active Revoked
- 2009-11-13 WO PCT/DE2009/001623 patent/WO2010054649A2/de active Application Filing
- 2009-11-13 DK DK09808976.6T patent/DK2403947T3/da active
- 2009-11-13 ES ES09808976.6T patent/ES2669018T3/es active Active
- 2009-11-13 PL PL09808976T patent/PL2403947T3/pl unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP2403947B1 (de) | 2018-02-14 |
| JP5930716B2 (ja) | 2016-06-08 |
| US20110293659A1 (en) | 2011-12-01 |
| WO2010054649A3 (de) | 2010-10-07 |
| DK2403947T3 (da) | 2018-05-28 |
| JP2012508697A (ja) | 2012-04-12 |
| EP2403947A2 (de) | 2012-01-11 |
| DE112009003267A5 (de) | 2012-11-15 |
| ES2669018T3 (es) | 2018-05-23 |
| DE102008057451A1 (de) | 2010-05-20 |
| WO2010054649A2 (de) | 2010-05-20 |
| PL2403947T3 (pl) | 2018-08-31 |
| US9885052B2 (en) | 2018-02-06 |
| CN102741414B (zh) | 2015-11-25 |
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