CN116440256A - 一种普适且高效的mRNA疫苗制备方法及产品 - Google Patents
一种普适且高效的mRNA疫苗制备方法及产品 Download PDFInfo
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- CN116440256A CN116440256A CN202310407516.7A CN202310407516A CN116440256A CN 116440256 A CN116440256 A CN 116440256A CN 202310407516 A CN202310407516 A CN 202310407516A CN 116440256 A CN116440256 A CN 116440256A
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Abstract
本发明公开了一种普适且高效的mRNA疫苗制备方法,得到抗原重组表达载体,转化细菌并发酵后,提取质粒并酶切获得抗原基因线性化转录模板,体外转录得到mRNA,加Cap1帽结构,经脂质纳米颗粒包封得到mRNA疫苗;所述重组表达载体包含如下依次连接的组合元件:T7启动子序列、人丙型肝炎病毒5’UTR截短序列、多克隆位点序列、人血红蛋白亚单位β基因3’UTR序列以及3个PolyA40串联序列,抗原基因序列插入在所述多克隆位点序列中;使用所述方法制备的狂犬病毒或带状疱疹病毒的mRNA疫苗在细胞内具有更稳定、更高的表达水平,免疫效价也更高,且具有较低的免疫原性。
Description
技术领域
本发明涉及生物医药技术领域,具体设计一种普适且高效的mRNA疫苗制备方法,还涉及该方法制备的mRNA疫苗。
背景技术
快速高效制备相应疫苗成为保障人们生命安全的重要途经。而mRNA疫苗的出现为解决这一难题带来了福音。该类疫苗主要目的是促使细胞合成目的蛋白,进而引起相应的免疫反应,实现机体保护。
mRNA疫苗主要由mRNA部分及其外部用来包裹的纳米制剂构成。其中,mRNA部分依次由维持mRNA稳定性的5’端的帽子结构、提升翻译效率的5’UTR区域、目的蛋白编码序列ORF、提升翻译效率且增强mRNA稳定性的3’UTR、及影响mRNA半衰期且维持其稳定的poly(A)尾巴这些结构组成。纳米制剂则主要指用来装载mRNA的脂质纳米颗粒,其成分主要包括脂质体、LNP、纳米脂质复合物、脂质多聚复合物等。该类制剂的主要功能为有效装载mRNA,防止mRNA降解及逃脱内涵体释放药物。
mRNA疫苗的制备方法相对简单,主要分为两部分,即设计并获得表达目的蛋白的具备上述结构的mRNA序列,其次将mRNA装载于纳米制剂中后就可完成mRNA疫苗的制备。
mRNA疫苗的优势在于其强烈的体液免疫和细胞免疫,具备良好的安全性,其表征参数可受控制。而该类疫苗最大的优势在于其研发周期短,具备快速从研发到生产的延期,可建立标准化具备普适性的平台和工艺,能够实现大量生产。
而在mRNA疫苗制备过程中的挑战主要为未经修饰的mRNA会诱导先天免疫反应,从而引起其他免疫反应。此外,mRNA疫苗的轻微不良反应比较常见。由于mRNA自身特点导致其在生理环境下不稳定,不正确的操作或者储存都将导致mRNA疫苗的失效。不同的纳米递送系统对于mRNA在细胞中的含量即其稳定性影响很大,而该类技术昂贵的原材料和极高的专利壁垒导致其制备工艺只掌握在少数人手中,致使因COVID-19而兴起的该技术无法在其他疫苗制备中获得更广泛的应用。
发明内容
有鉴于此,本发明的目的在于提供一种普适且高效的mRNA疫苗制备方法;该方法实现了mRNA疫苗稳定、高效的制备,使其在免疫过程中具备安全、高效的疫苗能力。基于此,本发明方法具有以下优点:1.目的蛋白编码序列中使用修饰核酸进行替换,提高mRNA稳定性的同时,降低先天免疫反应;2.筛选更优的UTR结构,使蛋白表达更稳定;3.筛选高效的5’端帽子添加技术,提高mRNA的纯度和稳定性;4.尾端长度及类型的优化,保证mRNA稳定的同时,实现病毒性mRNA的仿真设计;5.筛选更优的纳米递送制剂类型及其比例配方,防止mRNA降解,或逃脱内涵体提前释放药物。本发明的目的之二在于提供由所述方法制备得到的mRNA疫苗产品,如狂犬病毒的mRNA疫苗或带状疱疹病毒的mRNA疫苗或分泌型带状疱疹病毒的mRNA疫苗。
为达到上述目的,本发明提供如下技术方案:
1、一种普适且高效的mRNA疫苗制备方法,包含如下步骤:
构建抗原基因重组表达载体,转化细菌并发酵后,提取质粒并酶切获得抗原基因线性化转录模板,体外转录得到mRNA,加Cap1帽结构,经脂质纳米颗粒包封得到mRNA疫苗;所述重组表达载体包含如下依次连接的组合元件:T7启动子序列、人丙型肝炎病毒5’UTR截短序列、多克隆位点序列、人血红蛋白亚单位β基因3’UTR序列以及3个PolyA40串联序列,所述组合元件的核苷酸序列如SEQ ID NO.1所示;抗原基因序列插入在所述多克隆位点序列中。
本发明优选的,所述抗原基因为狂犬病毒基因或带状疱疹病毒基因或分泌型带状疱疹病毒基因;所述狂犬病毒基因的核苷酸序列如SEQ ID NO.3所示,所述带状疱疹病毒基因的核苷酸序列如SEQ ID NO.4所示,所述分泌型带状疱疹病毒基因的核苷酸序列如SEQID NO.5所示。
本发明优选的,其特征在于,所述重组表达载体的骨架为pIVT,所述细菌为Stbl3大肠杆菌。
本发明优选的,所述体外转录的反应体系中使用N1-甲基-假尿苷或假尿苷替换UTP。
本发明优选的,所述脂质纳米颗粒由DMAP-BLP、DSPC、cholesterol和DMG-PEG2000按摩尔比46.3:9:42.7:2配置,所述脂质纳米颗粒和mRNA的质量比为26.6:1。
2、由权利要求1~6任一项所述方法制备得到的mRNA疫苗。
本发明优选的,所述mRNA疫苗含有Cap1帽结构和N1-甲基-假尿苷。
本发明优选的,狂犬病毒的mRNA疫苗核苷酸序列如SEQ ID NO.8所示。
本发明优选的,带状疱疹病毒的mRNA疫苗核苷酸序列如SEQ ID NO.9所示。
本发明优选的,分泌型带状疱疹病毒的mRNA疫苗核苷酸序列如SEQ ID NO.10所示。
本发明的有益效果在于:
在本发明中,首先对mRNA的稳定性进行了相关设计与验证。为了模仿病毒mRNA自己身特点,选取了HCV的5’UTR部分区域,及HBB的3’UTR区域,用来增强mRNA的稳定,并且验证了5’UTR区域对于mRNA的动态变化至关重要。此外,经研究报道,修饰核苷酸的替换可以在提高mRNA稳定性的同时,降低先天免疫反应,因此,本发明筛选了Pseudouridine和N1-methyl-pseudouridine作为候选修饰核酸,经过实验验证,使用N1转录的mRNA在细胞内具备更高的表达水平。随后,确定了使用的纳米制剂类型及比例后,对其mRNA-LNP的包封率、稳定性、细胞中的表达状态进行检测,最终确定了整个mRNA-LNP的制备工艺。
待制备工艺确定后,本发明基于已发表的论文,选取了狂犬病毒及两种带状疱症病毒抗原蛋白作为研究对象,拟通过本发明方法,与已发表的实验数据进行比较,进而确定该方法所得的mRNA疫苗是否符合学术界的标准。其中,本发明中选取了市面上已流通的狂犬病毒灭活疫苗作为阳性对照一起开展相关实验。实验结果均显示,由本发明方法所制备的三种mRNA均能获得高效价的免疫反应。其中两种类型带状疱症病毒mRNA疫苗均能达到已发表文章中的免疫水平(Han Cao et al,Vaccines,2021),而利用本发明方法制备的狂犬病毒mRNA疫苗所获得的中和抗体滴度是参考文献中中和抗体滴度的5倍,并且在本发明中,使用较于文献中更低的mRNA疫苗量(Margit Schnee et al.PLOS Neglected TropicalDiseases,2016)。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为mRNA表达载体上各元件核酸理论序列;
图2为经改造的pIVT载体图谱;
图3为PNI仪器参数设置图;
图4为mRNA设计与验证(A,三种表达GFP mRNA载体设计示意图;B,三种设计下均能获得稳定mRNA体外转录产物;C,D2获得的GFP mRNA在293FT细胞中检测不到蛋白水平表达;D,细胞荧光镜检及流式分析均显示D1获得的GFP mRNA能获得高水平、高稳定的细胞蛋白水平表达;E,D1,D3不同设计中GFP阳性细胞比例统计结果展示。以上实验均至少独立重复2次,并展示2次独立重复实验结果。);
图5为最佳修饰核酸鉴定(A,本实验采用mRNA表达原件示意图;B,等量不同修饰核酸获得mRNA经琼脂糖凝胶电泳结果显示,N1组获得较大产量的mRNA;C,细胞实验及流式分析实验均显示,使用N1转录的mRNA在细胞内具备更高水平的表达。);
图6为mRNA-LNP稳定性检测;
图7为狂犬病毒抗原mRNA设计与表达验证(A,狂犬病毒抗原mRNA表达模型各元件示意图;B,经体外转录获得的mRNA琼脂糖电泳结果显示可获得稳定mRNA产物;C,狂犬病毒抗原mRNA转染进细胞后,经过抗体免疫荧光染色显示,该mRNA能够正常表达。);
图8为带状疱疹及分泌型带状疱疹病毒抗原mRNA设计与表达验证(A,带状疱疹及分泌型带状疱疹病毒抗原mRNA表达模型各元件示意图;B,经体外转录获得的mRNA琼脂糖电泳结果显示可获得稳定mRNA产物;C,带状疱疹及分泌型带状疱疹病毒抗原mRNA转染进细胞后,经过抗体免疫荧光染色显示,该mRNA能够正常表达。);
图9为小鼠免疫时间表;
图10为免疫小鼠血清中总IgG滴度检测(A,D38狂犬疫苗各组总IgG滴度检测结果显示,mRNA疫苗总IgG滴度高于市面上使用的灭活病毒疫苗;B,D38不同类型带状疱症病毒mRNA疫苗各组总IgG滴度结果显示,不论高低计量的不同类型带状疱症病毒mRNA疫苗均能诱导产生高水平的总IgG滴度。);
图11为不同采血点不同类型带状疱症病毒mRNA疫苗各种总IgG滴度检测;
图12为ELISpot检测免疫小鼠脾脏细胞中免疫因子分泌状况(A,D38狂犬病毒不同剂量及不同类型疫苗各中IFN-γ斑点数;B,D38狂犬病毒不同剂量及不同类型疫苗各中IL-2斑点数;C,D3带状疱症病毒不同剂量及不同类型疫苗各中IFN-γ斑点数;D,D38带状疱症病毒不同剂量及不同类型疫苗各中IL-2斑点数。);
图13为ICS检测免疫小鼠脾脏细胞中IFN-γ阳性T细胞比例(A,D38狂犬病毒不同剂量及不同类型疫苗各中CD4+、IFN-γ+细胞比例;B,D38狂犬病毒不同剂量及不同类型疫苗各中CD8+、IFN-γ+细胞比例;C,D3带状疱症病毒不同剂量及不同类型疫苗各中CD4+、IFN-γ+细胞比例;D,D38带状疱症病毒不同剂量及不同类型疫苗各中CD8+、IFN-γ+细胞比例。);
图14为D38狂犬病毒mRNA疫苗组血清中病毒中和抗体滴度。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好的理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1、mRNA表达骨架载体设计与克隆
各元件核酸理论序列为T7-HCVs 5’UTR-MCS-HBB 3’UTR-PolyA40x3_G(图1,SEQID NO.1)
其中,橘色部分表示T7启动子序列,深蓝色部分表示丙型肝炎病毒(HCVs)5’UTR截短序列,红色部分表示多克隆位点(MCS)序列,绿色部分表示人血红蛋白亚单位β(HBB)基因3’UTR序列,浅蓝色部分表示3个PolyA40串联序列。SEQ ID NO.1所示序列经人工合成后经Esp3I酶切连接到经改造的pIVT载体(SEQ ID NO.2,图2)上,得到mRNA表达重组载体pIVT-MCS。
HCV 5’UTR因其自带IRSE原件成为候选元件,同时为了尽量缩短5’UTR序列,也进行了截短测试,最终实验结果显示HCVs 5’UTR效果更佳;MCS为多克隆酶切位点,可在此处插入任何目的蛋白序列进行后续mRNA疫苗制备;HBB 3’UTR能够提升翻译效率并增强mRNA稳定性;PolyA40x3_G尾巴能够促进mRNA稳定性,延长半衰期,有效防止mRNA降解。
选择狂犬病毒抗原蛋白(KQ)mRNA表达核酸序列(SEQ ID NO.3)、带状疱疹病毒抗原蛋白(PZ)mRNA表达核酸序列(SEQ ID NO.4)和分泌型带状疱疹病毒抗原蛋白(gES)mRNA表达核酸序列(SEQ ID NO.5),以上序列在已发表文献中均已显示可以制备为mRNA疫苗,并产生相应的免疫反应。经专业公司合成后,经HindIII及XhoI酶切连接pIVT-MCS载体后,获得pIVT-KQ(用于表达狂犬病毒抗原蛋白mRNA),pIVT-PZ(用于表达带状疱症病毒抗原蛋白mRNA),及pIVT-gES(用于表达分泌型带状疱症病毒抗原蛋白mRNA)质粒,用于该表达载体普适性及高效性论证。将上述质粒转化进Stbl3菌株,用于后续的质粒制备。
实施例2、菌株发酵、质粒提取和纯化
一、细菌发酵
(1)以发酵体积5%确定初始Stbl3细菌培养体积,配置TB培养基;
(2)高压灭菌15min,冷却过夜后,以终浓度为100μg/ml浓度加入抗生素;
(3)37℃摇床恒温培养14-16h,转速200rpm,待OD600达到2.0-3.0后备用;
(4)将发酵罐进行pH校准,蠕动泵校正;
(5)根据发酵罐发酵体积配置TB培养基;
(6)培养基溶于发酵罐中,经高压灭菌15min,冷却备用;
(7)发酵罐装机后,运行过夜,检测灭菌是否完全;
(8)对发酵罐进行OD校准;
(9)以100μg/ml浓度加入抗生素后,取2ml培养基作为后续检测用阴性对照;
(10)按照发酵总体积的5%进行菌种接种,取1ml样品进行OD600检测;
(11)设定OD为30%,pH为7.0,温度为37℃,发酵12-14h。
二、细菌浓缩与透析换液
(1)用纯净水清洗切向流微滤套件;
(2)0.5M NaOH冲洗切向流微滤系统:调节蠕动泵泵速,使进口端压力为0.2bar,回流端和透过端阀门全开,循环冲洗30min,冲洗结束后,排空系统,收集碱液;
(3)纯水冲洗切向流微滤系统:调节蠕动泵泵速和回流端阀门,使进口端压力为0.2bar,回流端压力为0bar,透过端阀门全开,冲洗切向流微滤系统以去除系统内残留碱液,当回流端和透过端料液pH值均呈中性时,停止冲洗;
(4)测试中空纤维水通量:调节蠕动泵泵速和回流端阀门,使进口端压力为0.25bar,回流端压力为0.2bar,透过端阀门全开,记录1min时透过端收集的纯水体积,同时检测管道体积;
(5)排空系统,上样:取新鲜下罐的大肠杆菌收获液,打开蠕动泵,使系统内充满料液,先关闭透过端,使料液循环平衡中空纤维5min;
(6)菌体浓缩:调节蠕动泵泵速、回流端和透过端阀门,使进口端压力为1.2bar,回流端压力为0.4bar,透过端压力为0.4bar,开始计时,实时记录三个压力表读数,透过端收集料液体积等,可以根据透过端流速衰减情况调节蠕动泵泵速和两个阀门;
(7)透析换液:当达到要求的浓缩倍数后,开始换液(Lysis bufferⅠ),以与透过端相同的流速向样品瓶中泵入置换缓冲液,同时记录透过端收集料液体积,三个压力表读数,可根据实际情况在透过端取样检测电导,当透过端料液电导与置换缓冲液电导接近时,完成透析换液,停止实验;
(8)排空系统,收集样品瓶中的料液,可使用30-50ml置换缓冲液冲洗切向流微滤系统,回收滞留在系统中的大肠杆菌,收集液取样检测大肠杆菌滴度;
(9)纯水冲洗切向流微滤系统:调节蠕动泵泵速和回流端阀门,使进口端压力为0.2bar,回流端压力为0bar,透过端阀门全开,约冲洗系统30min,去除系统内残留的料液;
(10)0.5M NaOH冲洗切向流微滤系统:调节蠕动泵泵速,使进口端压力为0.2bar,回流端和透过端阀门全开,循环冲洗30min,冲洗结束后,排空系统,收集碱液;
(11)纯水冲洗切向流微滤系统:调节蠕动泵泵速和回流端阀门,使进口端压力为0.2bar,回流端压力为0.5bar,透过端阀门全开,冲洗切向流微滤系统以去除系统内残留的碱液,当回流端和透过端料液pH值均呈中性时,停止冲洗;
(12)测试中空纤维水通量:调节蠕动泵泵速和回流端阀门,使进口端压力为0.25bar,回流端压力为0.2bar,透过端阀门全开,记录1min时透过端收集的纯水体积;
(13)0.1M NaOH保存微滤中空纤维,拆卸切向流微滤系统,结束实验。
浓缩与透析参数:
浓缩倍数:6倍;平均流速33.281LMH(升/平方米/小时)
透析换液:5次;置换为裂解前缓冲液,平均流速28.389LMH(升/平方米/小时)
浓缩透析前湿重比4.2%,浓缩透析后湿重比24.5%。
三、细菌裂解:
(1)调节固含量:将经过收获后的菌液用Alkaline Lysis Solution I调节固含量至5%;
(2)加入等体积的Alkaline Lysis Solution II,颠倒混匀,时间控制在3min内;
(3)加入等体积的Alkaline Lysis Solution III,颠倒混匀,至溶液全部产生均一白色沉淀,冰上放置10min;
(4)将裂解后澄清与沉淀分离,弃沉淀,并在澄清液中加入CaCl2溶液,终浓度为0.5M。
裂解液配方:
Alkaline Lysis Solution I:50mM glucose,25mM Tris-Cl(pH8.0),10mM EDTA(pH 8.0)。在15psi下高压蒸汽灭菌,在4℃下储存,使用前加入RNA酶使终浓度为100ug/mL。
Alkaline Lysis Solution II:0.2M NaOH(以10M母液稀释后制备),1%(w/v)SDS,solution II溶液室温下现配现用。
Alkaline Lysis Solution III:5M醋酸钾60.0ml,冰醋酸11.5ml,H2O 28.5ml。所得溶液钾离子终浓度为3M,醋酸根离子终浓度为5M。将溶液储存在4℃下,并在使用前将其转移到冰浴条件下。
四、裂解液澄清
(1)润湿滤器:用缓冲液(或纯化水)对滤器进行表面润湿,润湿流速约7ml/min,润湿体积约100ml,排空滤器及管路;
(2)过滤:取实验用料液,采用恒流模式进行澄清过滤,过滤速度设定10-20ml/min左右,监控过滤压力和浊度;
(3)终止过滤:过滤过程中当浊度贯穿,或压力达到1bar左右,或样品滤完,即终止实验;
(4)顶洗滤器:根据实验需求,决定是否用缓冲液顶洗滤器和管路。
澄清参数:
澄清速度:平均流速751.6LMH(升/平方米/小时)
五、澄清液浓缩与置换
(1)清洁:测试用管路和接头,不锈钢夹具用纯化水冲洗,去除表面杂质;
(2)安装切向流过滤系统:连接蠕动泵,管路和接头,压力表,将超滤膜包安装到不锈钢夹具中,用扭力扳手旋紧;
(3)稳定化再生纤维素材质Hydrosart安装扭力是17Nm;
(4)用纯水冲洗超滤系统10min后,再用0.5M NaOH溶液冲洗切向流系统30min;
(5)纯化水冲洗切向流过滤系统至中性,测试水通量,测试参数为Pin=2.0bar,Pout=0.5bar,透过端全打开;
(6)排空切向流过滤系统,准备上样;
(7)回流端全打开,透过端关闭,采用Pin=0.8bar,Pout=0bar压力条件下先循环样品5min;随后缓慢打开透过端,调节泵速度、进口、回流端压力参数,由于浓缩过程中样品浓度逐渐增加,黏度增加,相同的泵动力,进口压力以及回流端压力随着浓度增加而升高,超滤到后期如果黏度较大,可调节泵动力进行控制进口压力不超过安全参数;
(8)超滤浓缩20倍;
(9)洗滤:采用DEAE纯化中的平衡液,进行8倍体积换液洗滤;
(10)顶洗:关闭透过端,用60ml顶洗Buffer分两次,每次30ml,并与上述样品收集在一起;
(11)顶洗结束后,打开透过端,在Pin=1.5bar,Pout=0.5bar左右的压力参数下继续用纯化水冲洗膜包5~10min,随后用0.5M NaOH清洗30min,NaOH清洗结束后,将其排空,再继续用纯化水把膜包冲洗至中性后再测试水通量,测试条件同使用前的测试条件;
(12)膜包保存:把膜包保存在0.1M NaOH中
澄清液浓缩与置换参数:
浓缩倍数:20倍,平均流速13.9LMH(升/平方米/小时)
透析换液:8次,置换为DEAE纯化前缓冲液,平均流速23.2LMH(升/平方米/小时)
六、质粒纯化
(1)纯化配置相应溶液:
Equilibration buffer:50mM Tris,10mM EDTA,pH 7.2;
Washing buffer:50mM Tris,10mM EDTA,0.6M NaCl,pH 7.2;
Elution buffer:50mM Tris,10mM EDTA,1M NaCl,pH 7.2;
(2)选用耗材为:CIMmultusTMDEAE 1mL Monolithic Column(Diethylamino)(2μmchannels)(311.5114-2);
(3)层析前处理:流速为2mL/min;0.5M NaOH,动态消毒20CV;纯化水洗10CV;层析柱体积为1ml,0.5M NaOH冲洗体积为20ml,纯化水冲洗体积为10ml;
(4)样品层析前调样:用去离子水稀释细菌裂解液至电导率为35-40mS/cm,再用0.45um过滤器过滤;
(5)CIMmultus DEAE层析:上样及冲洗流速:2CV/min;Δp:≤1.8Mpa;上样体积:0.5-1mg样品;
(6)配置相应buffer:
Equilibration buffer:50mM Tris,10mM EDTA,3M(NH4)2SO4,pH 7.2
Washing buffer:50mM Tris,10mM EDTA,1.7M(NH4)2SO4,pH 7.2;
Elution buffer:50mM Tris,10mM EDTA,0.4M(NH4)2SO4,pH 7.2;
Regeneration buffer:50mM Tris,10mM EDTA,pH 7.2;
(7)选用耗材为:CIMmultusTMC4 HLD 1mL Monolithic Column(HLD Butyl)(2μmchannels)(311.8136-2);
(8)层析前处理:流速为2mL/min;0.5M NaOH,动态消毒20CV;纯化水洗10CV;层析柱体积为1ml,0.5M NaOH冲洗体积为20ml,纯化水冲洗体积为10ml;
(9)样品层析前调样:调节液来自DEAE的含pDNA洗脱液,每1体积洗脱液加入3体积的4M(NH4)2SO4;
(10)CIMmultus C4 HLD层析:上样及冲洗流速:2CV/min;Δp:≤1.8Mpa;上样体积:0.5-1mg样品;
七、质粒浓缩与置换
此过程同上述“五、澄清液浓缩与置换”,具体步骤及参数参考“五、澄清液浓缩与置换”。
实施例3、三种疫苗mRNA制备与纯化
一、大规模线性化模板准备
(1)按照下列表1体系使用酶切线性化转录模板,若要获得更多线性化模板,体系可等比例扩大
表1酶切体系
(2)在恒温箱种放置3D旋转混合仪,设置转速为10rpm,37℃,反应时长应大于16h。
(3)所有反应体系应在RNase-Free环境中进行,采用纯度在99.5%级以上的乙醇,使用RNase-Free级别水进行相关稀释;
(4)将磁珠液提前30min从4℃冰箱中取出,平衡至室温;
(5)颠倒或漩涡震荡使磁珠充分混匀,吸取纯化模板0.5倍体积磁珠液,加入DNA样品中,使用移液器轻轻吹打10次混匀;
(6)室温孵育10min,使DNA结合至磁珠上;
(7)将样品置于磁力架上,静置5min,待溶液澄清后,小心移除上清,切勿将样品从磁力架上取下;
(8)保持样品始终处于磁力架上,加入初始样品体积1.5倍体积的新鲜配置的80%乙醇漂洗磁珠,室温孵育30s,小心移除上清;
(9)重复步骤(6);
(10)保持样品始终处于磁力架上,室温开盖干燥磁珠5min;
(11)将样品从磁力架上取出,加入适量无核酸酶水,使用移液器吹打充分混匀,室温静置2min;
(12)将样品放置于磁力架上,静置5min,待溶液澄清后,小心吸取上清至无核酸酶离心管中;
(13)16000g离心十分钟,取上清至无核酸酶离心管中,以完全去除残留的磁珠;
(14)取上清,Nano-drop测定浓度备用。
二、mRNA体外合成(IVT)
(1)按照下列表2配方配置反应体系,若要获得更多mRNA,体系可等比例扩大
表2mRNA体外合成体系
(2)将上述试剂放置于反应袋中,将反应袋安装于ReadyToProcessTMWAVE 25波浪式生物反应系统中,37℃,3h。
三、mRNA纯化
(1)反应结束后,取出反应袋,使用avant层析系统搭配多级纯化柱进行纯化;
(2)配置buffer:
Buffer A:50mM PB,250mM NaCl,2mM EDTA,pH 7.0;
Buffer B:50mM PB,1M NaCl,2mM EDTA,pH 7.0;
Buffer C:50mM PB,2mM EDTA,pH 7.0;
Buffer D:10mM Tris,pH 7.2。
(3)选用耗材为:CIMmultus Oligo dT18 C6-1(2)(cat#:311.1218-2);
(4)层析前处理:流速为2mL/min;0.5M NaOH,动态消毒10CV;纯化水洗10CV;层析柱体积为1ml,0.5M NaOH冲洗体积为10ml,纯化水冲洗体积为10ml;
(5)样品层析前调样:IVT反应后混合物,用Buffer B按照3:1比例将混合盐浓度调节至250mM;
(6)CIMmultus Oligo dT18层析:上样及冲洗流速:2CV/min;Δp:≤1.8Mpa;上样体积:0.5-1mg样品;
(7)配置buffer:
Buffer E:50mM Tris-HCl,3M NaCl,2mM EDTA,pH 7.2;
Buffer F:50mM Tris-HCl,2M NaCl,2mM EDTA,pH 7.2
Buffer G:50mM Tris-HCl,2mM EDTA,pH 7.2
(8)选用耗材为:CIMmultus C4 HLD-1(2)(cat#:311.8136-2);
(9)层析前处理:流速为2mL/min;0.5M NaOH,动态消毒20CV;纯化水洗10CV;层析柱体积为1ml,0.5M NaOH冲洗体积为20ml,纯化水冲洗体积为10ml;
(10)样品层析前调样:取CIMmultus Oligo dT18洗脱液按1:2比例加入BufferE溶液,将样品盐浓度调整至2M;
(11)CIMmultus C4 HLD层析:上样及冲洗流速:2CV/min;Δp:≤1.8Mpa;上样体积:0.5-1mg样品;
四、mRNA加帽反应
(1)将上述纯化产物经使用切向流系统浓缩置换至纯水(RNsae-Free)后,经65℃变性10min,迅速至于冰水混合物中降温后备用,此步骤旨在在打开5’端结构,提高加帽效率,具体反应体系如下表3:
表3mRNA加帽反应体系
(2)将上述试剂加入反应袋中,将反应袋安装于ReadyToProcessTMWAVE 25波浪式生物反应系统中,37℃,3h。
五、加帽后mRNA纯化
反应结束后取出反应袋,使用avant层析系统搭配多级纯化柱进行纯化,具体纯化步骤及参数同上述mRNA纯化步骤。纯化后用于LNP制备,需采用切向流系统将mRNA换液于25mM sodium citrate(pH 4.0)buffer中备用。
实施例4、三种疫苗mRNA-LNP制备
一、脂质纳米颗粒(LNP)制备
(1)试剂准备:按照下列表4使用乙醇配制各lipid储存液,分装后可置于-80℃长期保存。
表4lipid储存液
成分 | 储存液浓度(mM) |
DMAP-BLP | 149.068323 |
DSPC | 15.8198 |
Cholesterol | 36.9585 |
DMG-PEG 2000 | 39.8533 |
(2)以0.5ml lipid混合液为例,按照下表5进行配置,其配比可等比放大:
表5lipid混合液体系
二、三种疫苗mRNA-LNP配置
(1)将溶解在RNA储存液中的mRNA母液通过超滤管进行液体交换,选取适当体积的30k超滤管,将mRNA母液加入管中,16000g,4℃离心10min,加入适当体积25mM柠檬酸钠缓冲液;
(2)16000g,4℃离心10min,加入适当体积25mM柠檬酸钠缓冲液,保证mRNA终浓度为167μg/ml,改液体交换时mRNA回收率接近100%,可按照初始质量进行25mM柠檬酸钠缓冲液体积计算;
(3)取5ml mRNA溶液加入至5ml BD注射器中;
(4)取2ml lipid混合液加入至1ml BD注射器中;
(5)按图3所示参数设置PNI仪器,并获取mRNA-LNP混合液。
(6)向制备好的mRNA-LNP混合液中加入PBS溶液至总体积为15ml;
(7)将步骤(6)中获得PBS稀释液加入超滤管中,20000g,25℃,10min,共离心三次,每次离心结束后用枪吹打超滤膜,直至溶液体积浓缩至稀释前体积,即约1.5ml;
(8)大体积mRNA-LNP混合液应采用切向流系统进行浓缩;若需冷冻保存,则需将mRNA-LNP混合液换液至含8%蔗糖的PBS溶液中。
实施例5、不同组合元件生成的mRNA稳定性验证
根据已发表的文献,包括Narayanasamy Elango,et.al,Optimized transfectionof mRNA transcribed from a d(A/T)100tail-containing vector Biochemical andBiophysical Research Communications,2005;Silke Holtkamp,et.al,Modification ofantigen-encoding RNAincreases stability,translational efficacy,and T-cellstimulatory capacity of dendritic cells,GENE THERAPY,2006;NicholasJ.McGlincy,et.al,Transcriptome-wide measurement of translation by ribosomeprofiling,Methods,2017;Kimberly J.Hassett,et.al,Optimization of LipidNanoparticles for Intramuscular Administration of mRNA Vaccines MolecularTherapy:Nucleic Acids,2019;Alexandra G.Orlandini von Niessen,et.al,ImprovingmRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3’UTRsIdentified by Cellular Library Screening,Molecular Therapy,2019.本申请筛选出了HCV 5’UTR全序列(SEQ ID NO.6),HCVs 5’UTR截短序列(SEQ ID NO.1所示序列的21~70bp所),HBB 3’UTR(SEQ ID NO.1所示序列的162~427bp)作为后续骨架载体的候选。基于此,本申请就筛选所得的各元件做相应设计(图4,A)。随后,就上述不同设计,本申请将GFP基因序列(SEQ ID NO.7)引入MCS区域(SEQ ID NO.1所示序列的71~161bp),并进行相关细胞实验用以验证不同设计对于mRNA表达及其稳定性的影响。
实验结果显示,采用上述3种设计均能在体外获得稳定的mRNA产物(图4,B),然而Design 2(后续简称D2)获得的mRNA在进行细胞水平蛋白表达验证实验时显示,通过荧光显微镜及流式分析均指出,该设计组合不能促使GFP在293FT细胞中表达(图4,C)。与此同时本申请对其他两组设计所获得mRNA进行同样的细胞实验室,结果显示,Design 1(后续简称D1)载体中的GFP在293FT细胞中能获得高水平、稳定的表达状态(图4,D,E)。综上所述,在mRNA表达载体设计中,HCVs 5’UTR和HBB 3’UTR同时存在才能保证mRNA的表达更加高效及稳定。因此,本申请后续选择Design 1的元件组合,即实施例1中T7-HCVs 5’UTR-MCS-HBB3’UTR-PolyA40x3_G(图1,SEQ ID NO.1)。
实施例6、mRNA中饰核酸鉴定
本发明对体外转录获得mRNA中所添加修饰核酸进行鉴定。基于GFP的表达实验显示,在mRNA体外转录过程中,添加N1-甲基-假尿苷组(N1-methyl-pseudouridine)不仅能获得较高产量的mRNA(图5,B),也能促使其转录的mRNA在293FT细胞内具备更高的表达水平(图5,C)。
实施例7、mRNA-LNP稳定性检验结果
本发明对mRNA-LNP的稳定性进行了检验,本发明使用新鲜制备的mRNAGFP-LNP分别进行细胞感染及分装储存。实验结果显示,在-80℃分别储存2天、15天、30天后,室温解冻并感染细胞,检测GFP表达情况。实验结果显示,不论冻存多久,其对293FT细胞的感染情况、mRNA的浓度、及包封率均无明显差异(图6,表5)。该实验说明了本发明方法所获得的mRNA-LNP具备高感染效率及高稳定性。
表5mRNA-LNP中mRNA浓度及包封率检测
实施例8、狂犬病毒、带状疱症及分泌型带状疱疹病毒制备的mRNA疫苗稳定性及表达情况
本发明分别选取了已经有文献报道的狂犬病毒、带状疱症及分泌型带状疱疹病毒三种病毒抗原蛋白作为测试对象。首先,采用实施例1中论证的mRNA表达元件组合构建表达载体(图7,A或图8,A),然后检测其mRNA稳定性及在细胞中的表达情况。实验结果显示,无论是狂犬病毒抗原蛋白,还是两种不同的带状疱疹抗原蛋白,均能获得稳定状态的mRNA(图7,B或图8,B),并且该mRNA在293FT细胞中均能正常表达,其中由于gES会促使其翻译后的蛋白分泌到细胞外,因此细胞中gES蛋白检测到的表达水平较低(图7,C或图8,C)。该实验说明,本发表所设计的mRNA体外表达模型具备普适性。
狂犬病毒、带状疱症及分泌型带状疱疹病毒制备的mRNA疫苗序列如SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10所示。
实施例9、动物实验验证mRNA疫苗的效能水平
本发明将获得的mRNA-LNP进行动物实验,评价在本发明技术方法下获得的mRNA疫苗的效能水平。具体实验安排如下:在该实验中,分别在给药前1天(D0)进行小鼠称重及采血,给药后14天(D15)采血,第1次给药后21天追加第2次给药,并在第2次给药后7天(D29)采血,第2次给药后16天(D38)采血并结束免疫,通过采取免疫动物脾脏等组织,进行相关免疫鉴定(图9)。
对图8中所述最终采血点所采血清中的个疫苗抗体滴度(titer)进行检测。实验结果显示,注射2针不同质量狂犬糖蛋白mRNA疫苗(KQ)及灭活病毒疫苗(PC)均能诱导小鼠产生高剂量总IgG,其中5μg mRNA疫苗组总IgG titers最高,并且mRNA疫苗总IgG滴度高于市面上使用的灭活病毒疫苗(图10,A)。同时,注射2针不同质量全序列疱疹糖蛋白(PZ)及分泌型疱疹糖蛋白(gES)mRNA均能诱导小鼠产生高剂量总IgG,其中5μg mRNA疫苗组总IgGtiters最高(图10,B)。
本发明选取了带状疱症病毒在不同采血点进行其对应血清中IgG总滴度的检测。实验结果显示,除阴性对照组外,其余各组均产生高剂量总IgG titers,其中5μg mRNA疫苗组总IgG titers上升速度及产生IgG均最高(图11)。
此外,本发明还对免疫小鼠脾脏细胞中各免疫因子分泌状况进行了检测。经2针疫苗注射后,于D38收取免疫小鼠脾脏,经一些列处理后获取脾脏细胞,并将3x105个脾脏细胞种于96孔板中,经狂犬病毒灭活疫苗及带状疱症肽库刺激后,进行IFN-γ及IL-2染色,并计阳性细胞数量,获得相应斑点数。结果显示,在所测试的3种疫苗中,无论是IFN-γ阳性斑点数,还是IL-2阳性斑点数,5μg mRNA疫苗组均具备最多斑点数,并且狂犬病毒mRNA疫苗诱导产生的IFN-γ及IL-2阳性斑点数均高于市面上使用的灭活病毒疫苗(图12,A,B)。同样的,在带状疱症病毒实验组中,无论是IFN-γ阳性斑点数,还是IL-2阳性斑点数,5μg疱疹糖蛋白全序列(PZ)mRNA疫苗组均具备最多斑点数(图12,C,D)。
此外,在本发明中还对免疫小鼠脾脏细胞中IFN-γ阳性T细胞比例进行了检测。经2针疫苗注射后,于D38收取免疫小鼠脾脏,经一些列处理后获取脾脏细胞,并将1x105个脾脏细胞种于96孔板中,经狂犬灭活疫苗及带状疱疹肽库刺激后,利用药物阻断IFN-γ分泌,进行CD3、CD4、CD8、及IFN-γ染色后,进行流式分析。其中CD3为脾脏中T细胞标记物。在CD3+分群下,分别检测CD4+;IFN-γ+及CD8+;IFN-γ+细胞。结果显示,不论是mRNA疫苗还是已投入使用的是灭活病毒疫苗(PC),均能产生较高比例IFN-γ阳性T细胞(图13,A,B)。同时,在带状疱症实验组中,mRNA疫苗同样能够刺激产生较高比例的IFN-γ阳性T细胞(图13,C,D)。
在本发明中,也检测了狂犬病毒mRNA疫苗在免疫小鼠中产生的中和抗体滴度。实验结果显示,无论是1μg mRNA疫苗组,还是5μg mRNA疫苗组,均能在38天诱导产生高剂量中和抗体。其中5μg mRNA疫苗组所诱导产生的中和抗体剂量最高(图14)。WHO认为免疫后血清中的病毒中和抗体≥0.5IU/ml即达到有效保护水平,并且在使用本发明技术制备的mRNA疫苗进行免疫后,其所获得的中和抗体滴度是本发明所采用的狂犬病毒抗原蛋白来源的mRNA疫苗参考文献中中和抗体滴度的5倍,并且在本发明中,使用较于文献中更低的mRNA疫苗量(Margit Schnee et al.PLOS Neglected Tropical Diseases,2016),说明本发明方法具备高效性。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (10)
1.一种普适且高效的mRNA疫苗制备方法,其特征在于,包含如下步骤:构建抗原基因重组表达载体,转化细菌并发酵后,提取质粒并酶切获得抗原基因线性化转录模板,体外转录得到mRNA,加Cap1帽结构,经脂质纳米颗粒包封得到mRNA疫苗;所述重组表达载体包含如下依次连接的组合元件:T7启动子序列、人丙型肝炎病毒5’UTR截短序列、多克隆位点序列、人血红蛋白亚单位β基因3’UTR序列以及3个PolyA40串联序列,所述组合元件的核苷酸序列如SEQ ID NO.1所示;抗原基因序列插入在所述多克隆位点序列中。
2.根据权利要求1所述的方法,其特征在于,所述抗原基因为狂犬病毒基因或带状疱疹病毒基因或分泌型带状疱疹病毒基因;所述狂犬病毒基因的核苷酸序列如SEQ ID NO.3所示,所述带状疱疹病毒基因的核苷酸序列如SEQ ID NO.4所示,所述分泌型带状疱疹病毒基因的核苷酸序列如SEQ ID NO.5所示。
3.根据权利要求1所述的方法,其特征在于,所述重组表达载体的骨架为pIVT,所述细菌为大肠杆菌Stbl3。
4.根据权利要求1所述的方法,其特征在于,所述体外转录的反应体系中使用N1-甲基-假尿苷或假尿苷替换UTP。
5.根据权利要求1所述的方法,其特征在于,所述脂质纳米颗粒由DMAP-BLP、DSPC、cholesterol和DMG-PEG 2000按摩尔比46.3:9:42.7:2配置,所述脂质纳米颗粒和mRNA的质量比为26.6:1。
6.由权利要求1~6任一项所述方法制备得到的狂犬糖蛋白mRNA疫苗或疱疹糖蛋白mRNA疫苗或分泌型疱疹糖蛋白mRNA疫苗。
7.根据权利要求6所述的疫苗,其特征在于,所述狂犬糖蛋白mRNA疫苗的核苷酸序列如SEQ ID NO.8所示。
8.根据权利要求6所述的疫苗,其特征在于,所述疱疹糖蛋白mRNA疫苗的核苷酸序列如SEQ ID NO.9所示。
9.根据权利要求6所述的疫苗,其特征在于,所述分泌型疱疹糖蛋白mRNA疫苗的核苷酸序列如SEQ ID NO.10所示。
10.根据权利要求6所述的疫苗,其特征在于,所述mRNA疫苗含有Cap1帽结构和N1-甲基-假尿苷。
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