CN117821482B - 一种编码猴痘病毒和新冠病毒融合抗原的mRNA疫苗 - Google Patents
一种编码猴痘病毒和新冠病毒融合抗原的mRNA疫苗 Download PDFInfo
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Abstract
本发明公开了一种mRNA分子，所述mRNA分子含有编码猴痘病毒M1R抗原的多核苷酸和编码新型冠状病毒RBD抗原的多核苷酸，以及，进一步地含有编码猴痘病毒A35R抗原的多核苷酸，本发明还公开了所述的mRNA分子在制备猴痘病毒病或新型冠状病毒病mRNA疫苗中的应用。本发明提供的编码融合抗原的mRNA疫苗，相比单独编码对应抗原的mRNA疫苗，能够针对猴痘和新冠诱导产生相当甚至更高水平的抗体反应，并对鼠痘病毒致死攻毒提供100%的免疫保护。所述疫苗需合成单个mRNA分子进行脂质体纳米颗粒包装，相比多价mRNA疫苗组合物具有更广阔的应用前景。
Description
技术领域
本发明属于生物工程技术领域，具体而言，涉及针对猴痘和新冠病毒的mRNA疫苗。
背景技术
在新冠病毒长期流行的背景下，其他新发突发传染病的出现不断对人类产生新的威胁。正痘病毒包括天花病毒、猴痘病毒、痘苗病毒和鼠痘病毒等，具有非常接近的进化距离。虽然当下猴痘病毒主要局限于某些特定人群，但新冠病毒感染引起的免疫功能失调可能会导致猴痘病毒易感人群的扩大，目前已出现新冠病毒和猴痘病毒共同感染的病例。因此有必要针对猴痘病毒和新冠病毒开发单组分融合疫苗，在产生正痘病毒免疫反应的同时，能够以加强免疫的方式增强对新冠病毒的免疫防护。
S蛋白是新冠病毒最为重要的保护性抗原，是重组新冠疫苗最核心的组分。相比之下猴痘病毒具有更为复杂的抗原谱，目前多数重组猴痘疫苗采取的是多价策略，针对M1R、A35R、H3L、A29L、B6R、E8L等不同抗原进行组合设计，构成多组分疫苗。mRNA疫苗具有可快速合成、免疫原性高等技术优势。本发明的目的就是基于mRNA技术，提供一种编码单组份融合抗原的mRNA疫苗，能够同时诱导产生针对猴痘和新冠抗原的高水平抗体反应。
发明内容
基于上述目的，本发明首先提供了一种mRNA分子，所述mRNA分子含有编码猴痘病毒M1R抗原的mRNA和编码新型冠状病毒RBD抗原的mRNA。
在一个优选的实施方案中，所述mRNA分子编码的多肽的序列如SEQ ID NO: 2。
在一个更为优选的实施方案中，所述mRNA分子编码的多核苷酸的序列如SEQ IDNO: 1所示。
在本发明的另一个优选的实施方案中，所述mRNA分子还含有编码猴痘病毒A35R抗原的mRNA。
在一个更为优选的实施方案中，所述mRNA分子编码的多肽的序列如SEQ ID NO:4。
更为优选地，所述mRNA分子编码的多核苷酸的序列如SEQ ID NO: 3所示。
在本发明的一个更为优选的实施方案中，所述mRNA分子的5’端还含有启动子、5’UTR，所述mRNA的3’端还含有3’UTR、终止密码子、poly(A)、BspQI酶切位点串联。
尤为优选地，所述启动子的序列如SEQ ID NO:5所示，所述5’UTR的序列如SEQ IDNO: 6所示，所述3’UTR的序列如SEQ ID NO: 7所示，所述终止密码子的序列如TGATAATAG所示，所述BspQI酶切位点串联的序列如GAAGAGC所示，所述poly(A)为110个核苷酸长度。
在一个优选的实施方案中，所述mRNA分子的序列如SEQ ID NO: 14或SEQ ID NO:15所示。
在一个更为优选的实施方案中，在所述mRNA分子的5’端还含有Cap1帽子结构。
其次，本发明提供了一种包裹上述的mRNA分子的脂质体纳米颗粒。
再次，本发明提供了一种上述的脂质体纳米颗粒的制备方法，所述方法包括以下步骤：
（1）以十七烷-9-基-8-（（2-羟乙基）（6-氧代-6-（（十一烷氧基）己基）氨基）辛酸酯、二硬脂酰基磷脂酰胆碱、甲氧基聚乙二醇二肉豆蔻酰甘油、胆固醇、按照50：10：1.5：38.5的摩尔比形成脂质混合物并制备溶有所述mRNA的溶液；
（2）将步骤（1）获得的脂质混合物与所述的mRNA的溶液混合。
在一个更为优选的实施方案中，步骤（2）中脂质混合物与mRNA的溶液的质量比例为1:3。
最后，本发明提供了一种上述mRNA在制备猴痘病毒病或新型冠状病毒病mRNA疫苗中的应用。
本发明提供的编码融合抗原的mRNA疫苗，相比单独编码对应抗原的mRNA疫苗，能够针对猴痘和新冠诱导产生相当甚至更高水平的中和抗体反应，并对鼠痘病毒致死攻毒提供100%的免疫保护。此外单组份融合mRNA疫苗制备简单，仅需合成单个mRNA分子进行脂质体纳米颗粒包装，相比多价mRNA疫苗组合物具有更广阔的应用前景。
附图说明
图1. mRNA合成模板质粒PUC57-tPA-M1Recto示意图；
图2. mRNA合成模板质粒PUC57- tPA-RBD示意图；
图3. mRNA合成模板质粒PUC57- tPA-A35Recto示意图；
图4. mRNA合成模板质粒PUC57- tPA-M1Recto-RBD示意图；
图5. mRNA合成模板质粒PUC57- tPA-M1Recto-dRBD-A35Recto示意图；
图6. 毛细管电泳表征合成的mRNA分子；
图7. 动态光散射表征经脂质体纳米颗粒包装的mRNA疫苗；
图8. mRNA侯选疫苗的特异性抗体水平；
图9. mRNA侯选疫苗的新冠假病毒中和抗体水平；
图10. mRNA侯选疫苗对鼠痘致死攻毒的免疫保护水平。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的权利要求所限定的保护范围构成任何限制。
实施例1. mRNA疫苗的制备
（1）构建模板质粒
tPA信号肽分别融合于编码抗原M1Recto、RBD、A35Recto、M1Recto-RBD、M1Recto-dRBD-A35Recto的N端，经序列优化，得到的目的核苷酸序列分别为SEQ ID NO: 8、SEQ ID NO: 9、SEQ ID NO: 10、SEQ ID NO: 1、SEQ ID NO: 3。按照5’->3’方向，将T7启动子（SEQ ID NO:5）、5’UTR（SEQ ID NO: 6）、目的核苷酸、3’UTR（SEQ ID NO: 7）、终止密码子（TGATAATAG）、110个nt的poly(A)序列、BspQI酶切位点串联（GAAGAGC），克隆入PUC57质粒获得模板质粒（图1-图5）。
（2）模板质粒线性化
200ul反应体系中，包含20μg模板质粒、10μl BspQI酶（10U/μl）、20μl 10×BspQ IBuffer和Nuclease-FreeH2O ，50℃反应1小时。酚氯仿抽提纯化线性化质粒，向DNA溶液中加入等体积酚氯仿（Tris饱和苯酚：氯仿：异戊醇=25:24:1）充分混匀；室温，12000g，离心10min，小心吸取上层水相，加入等体积氯仿溶液（氯仿：异戊醇=24:1），充分混匀；同上离心后小心吸取上清，检测DNA浓度。
（3）mRNA体外转录及纯化
对编码M1Recto、RBD、A35Recto、M1Recto-RBD、M1Recto-dRBD-A35Recto的mRNA分子的体外转录，N1-甲基假尿苷对尿嘧啶的修饰比例均为100%。100ul反应体系中，包含5μg线性化质粒、10μl T7 RNA Polymerase（50U/μl）、5μl无机焦磷酸酶（0.1U/μl）、5μl RNaseInhibitor（40U/μl）、10μl 10×Reaction buffer、10μl ATP（100mM）、10μl GTP（100mM）、10μl m1ψ/UTP（100mM）、10μl CTP（100mM）和Nuclease-Free H2O （上述试剂购自诺唯赞公司），混合均匀后37℃反应2小时。随后在反应体系中加入5μlDNase I（1U/μl），37℃反应15min，去除转录DNA模板。如上使用酚氯仿纯化mRNA转录产物。
（4）mRNA加帽及纯化
100ul反应体系中，包含200ug转录mRNA、50μl 10 x Capping Reaction buffer、25μl GTP（10 mM）、25μl SAM（4 mM）、25μl Vaccinia Capping Enzyme（10U/μl）、25μl2'-O-Methyltransferase（50U/μl）和Nuclease-Free H2O（上述试剂购自诺唯赞公司），混合均匀后37℃反应1小时，如上使用酚氯仿纯化mRNA转录产物。使用毛细管电泳检测mRNA转录产物的分子完整性（图6），结果表明制备出的mRNA-M1Recto（SEQ ID NO:11）、mRNA-RBD（SEQ IDNO:12）、mRNA-A35Recto（SEQ ID NO:13）、mRNA-M1Recto-RBD（SEQ ID NO:14）、 mRNA-M1Recto-dRBD-A35Recto（SEQ ID NO:15）产物大小符合预期，纯度达到90%以上。
（5）mRNA脂质体纳米颗粒包装
SM-102（十七烷-9-基-8-（（2-羟乙基）（6-氧代-6-（（十一烷氧基）己基）氨基）辛酸酯），购自赛诺邦格公司）、DSPC（二硬脂酰基磷脂酰胆碱，购自赛诺邦格公司）、DMG-PEG2000（甲氧基聚乙二醇二肉豆蔻酰甘油、购自赛诺邦格公司）、胆固醇（购自艾伟拓公司）按照50：10：1.5：38.5的摩尔比溶解于乙醇，配置乙醇相；mRNA分子溶解于50 mM乙酸钠缓冲液（pH5.0），配置水相。微流控包装时，乙醇相和水相的体积比为1:3，总流速为12 ml/min。包装后超滤浓缩换液至PBS缓冲液，检测包封率和有效浓度后4°C 保存。动态光散射检测表明，脂质体纳米颗粒包装的mRNA疫苗具有均一的粒径分布（图7），LNP-mRNA-M1Recto、LNP-mRNA-RBD、LNP-mRNA-A35Recto、LNP-mRNA-M1Recto-RBD、LNP-mRNA-M1Recto-dRBD-A35Recto的平均直径分别为74.40nm、73.82nm、73.27nm、85.91nm、83.93nm，分散系数均小于0.05。
实施例2. mRNA疫苗的免疫反应
在BALB/c小鼠模型中，使用肌肉注射的方式，在第0天和第14天分别接种5μg的mRNA-M1Recto、mRNA-RBD、mRNA-A35Recto、mRNA-M1Recto-RBD、mRNA-M1Recto-dRBD-A35Recto候选疫苗（每组6只），第14天和28天采血取血清，检测特异性IgG抗体及中和抗体水平，并在28天进行鼠痘致死攻毒。
（1）特异性IgG抗体反应
将M1R（义翘神州，40904-V07H）、A35R（义翘神州，40886-V07E ）、RBD（义翘神州，40592-V08H136）重组蛋白稀释至1μg/ml的浓度，过夜包被于96孔板，封闭后通过酶联免疫吸附法检测IgG抗体效价，用two-way ANOVA with Šidák’s multiple comparison test进行统计分析。
针对M1R的特异性抗体反应，mRNA-M1Recto-RBD和mRNA-M1Recto-dRBD-A35Recto单次免疫后14天，IgG抗体效价几何平均值分别为19454和7798，加强免疫后14天（首次免疫后28天）抗体效价显著提升，几何平均值分别为3647529和486407，为mRNA-M1Recto抗体效价（162181）的20倍（p值小于0.0001）和3倍。
针对RBD的特异性抗体反应，mRNA-M1Recto-RBD和mRNA-M1Recto-dRBD-A35Recto单次免疫后14天， IgG抗体效价几何平均值均为16218，加强免疫后14天（首次免疫后28天）抗体效价显著提升，几何平均值分别为2023019和1093956，为mRNA-RBD抗体效价（145881）的14倍（p值小于0.0001）和7倍（p值等于0.0002）。
针对A35R的特异性抗体反应，mRNA-M1Recto-dRBD-A35Recto加强免疫后28天的IgG抗体效价几何平均值为1500，与mRNA-A35Recto相比无统计差异，但显著高于PBS对照组（p值小于0.0001）。（图8）
（2）新冠假病毒中和抗体反应
通过稳转人ACE2的293细胞感染新冠假病毒，检测加强免疫后14天（首次免疫后28天）的血清新冠假病毒中和抗体水平。针对新冠XBB 1.16变异株假病毒，mRNA-M1Recto-RBD和mRNA-M1Recto-dRBD-A35Recto的新冠假病毒中和抗体效价几何平均值达到480和1694，分别为mRNA-RBD中和抗体效价（277）的2倍和6倍（p值等于0.027）。针对新冠EG.5.1变异株假病毒，mRNA-M1Recto-RBD和mRNA-M1Recto-dRBD-A35Recto的新冠假病毒中和抗体效价几何平均值达到251和1714，分别为mRNA-RBD中和抗体效价（115）的2倍（无统计学差异）和15倍（p值等于0.0004）。上述结果表明mRNA-M1Recto-RBD激活产生的新冠中和抗体水平与mRNA-RBD相当，而mRNA-M1Recto-dRBD-A35Recto相比mRNA-RBD能够诱导产生更高水平的新冠中和抗体（图9）。
（3）鼠痘病毒致死攻毒保护性
通过BS-C-1细胞感染，扩增培养鼠痘病毒（ATCC VR-1374）。首次免疫后28天，每只小鼠腹腔攻毒200 PFU鼠痘病毒，18天内监测小鼠存活情况，用Log-rank (Mantel-Cox)test 进行统计分析。在一次致死攻毒实验中，mRNA-M1Recto-RBD和mRNA-M1Recto-dRBD-A35Recto免疫组的存活率均为100%，相比PBS对照组（存活率0%）具有显著的免疫保护性（p值等于0.0009）。上述结果证实编码M1Recto-RBD和M1Recto-dRBD-A35Recto的mRNA疫苗，能够对正痘病毒的致死攻毒提供完全的免疫保护（图10）。
Claims (12)
1. 一种mRNA分子，其特征在于，所述mRNA分子编码的多肽的序列如SEQ ID NO: 2所示。
2. 根据权利要求1所述的mRNA分子，其特征在于，所述mRNA分子的多核苷酸的序列如SEQ ID NO: 1所示。
3. 一种mRNA分子，其特征在于，所述mRNA分子编码的多肽的序列如SEQ ID NO: 4所示。
4. 根据权利要求3所述的mRNA分子，其特征在于，所述mRNA分子的多核苷酸的序列如SEQ ID NO: 3所示。
5.根据权利要求1-4任一所述的mRNA分子，其特征在于，所述mRNA分子的5’端还含有启动子、5’UTR，所述mRNA分子的3’端还串联有3’UTR、终止密码子、poly(A)、BspQI酶切位点。
6.权利要求5所述的mRNA分子在制备猴痘病毒病或新型冠状病毒病mRNA疫苗中的应用。
7. 根据权利要求5所述的mRNA分子，其特征在于，所述启动子的序列如SEQ ID NO:5所示，所述5’UTR的序列如SEQ ID NO: 6所示，所述3’UTR的序列如SEQ ID NO: 7所示，所述终止密码子的序列如TGATAATAG所示，所述BspQI酶切位点串联的序列如GAAGAGC所示，所述poly(A)为110个核苷酸长度。
8. 根据权利要求7所述的mRNA分子，其特征在于，所述mRNA分子的序列如SEQ ID NO:14或SEQ ID NO: 15所示。
9.根据权利要求8所述的mRNA分子，其特征在于，在所述mRNA的5’端还连接有Cap1帽子结构。
10.一种包裹权利要求9所述的mRNA分子的脂质体纳米颗粒。
11.权利要求10所述的脂质体纳米颗粒的制备方法，其特征在于，所述方法包括以下步骤：
（1）以十七烷-9-基-8-（（2-羟乙基）（6-氧代-6-（（十一烷氧基）己基））氨基）辛酸酯、二硬脂酰基磷脂酰胆碱、甲氧基聚乙二醇二肉豆蔻酰甘油、胆固醇按照50：10：1.5：38.5的摩尔比形成脂质混合物并制备溶有权利要求7所述mRNA的溶液；
（2）将步骤（1）获得的脂质混合物与所述的mRNA的溶液混合。
12.根据权利要求11所述的方法，其特征在于，步骤（2）中脂质混合物与mRNA的溶液的质量比例为1:3。
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