WO2023143600A1 - 一种用于核酸递送的新型可电离脂质及其lnp组合物和疫苗 - Google Patents
一种用于核酸递送的新型可电离脂质及其lnp组合物和疫苗 Download PDFInfo
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- WO2023143600A1 WO2023143600A1 PCT/CN2023/073789 CN2023073789W WO2023143600A1 WO 2023143600 A1 WO2023143600 A1 WO 2023143600A1 CN 2023073789 W CN2023073789 W CN 2023073789W WO 2023143600 A1 WO2023143600 A1 WO 2023143600A1
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- lipid
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- cationic lipid
- cationic
- mrna
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Abstract
本发明提供了一种新型阳离子脂质和脂质纳米颗粒及核酸疫苗。本发明选择特定的阳离子脂质制备的脂质纳米颗粒mRNA疫苗,发现其较现有技术具有更好的体外稳定性以及免疫原性。
Description
本发明涉及生物医药技术领域,具体涉及一种用于核酸递送的新型可电离脂质及其LNP组合物和疫苗。
目前递送mRNA在临床上经过验证的系统为脂质纳米粒(Lipid Nanoparticle,LNP),属于脂质形成的纳米微粒,其中原理包括阳离子脂质,而现有技术研究表明,mRNA递送至细胞内后,mRNA表达率偏低,例如Dlin-MC3-DMA作为阳离子脂质构建LNP,mRNA的表达量为0.63%(Maugeri,Marco et al.“Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells.”Nature Communications,2019),因此,阳离子脂质的结构是影响mRNA表达量的关键因素。
流感疫苗是用来预防流行性感冒病毒引起的流行性感冒(简称流感)的疫苗,适用于任何可能感染流感病毒的健康人,每年在流行季节前接种一次,免疫力可持续一年。
流感疫苗是预防和控制流感的主要措施之一。接种流感疫苗可以减少接种者感染流感的机会或者减轻流感症状。
2018年2月22日世界卫生组织发布的2018-2019年度北半球季节性流感疫苗推荐组分,四价流感病毒裂解疫苗包含甲型H1N1、甲型H3N2、乙型Yamagata系(By)、乙型Victoria系(Bv)四种流感病毒抗原成分,三价流感病毒裂解疫苗包含甲型H1N1、甲型H3N2、乙型Victoria系(Bv)三种流感病毒抗原成分,不包括乙型Yamagata系(By)流感病毒抗原成分。四价流感病毒裂解疫苗包含上述两种甲型和两种乙型共四种流感病毒抗原成分,可涵盖更多的流感流行型别,将有效预防和控制流感疫情。
目前已上市的流感疫苗主要为全病毒灭活疫苗、裂解疫苗和亚单位疫苗,尚无mRNA疫苗。
发明内容
本发明术语“中性脂质”术语是指不带电荷的、非磷酸甘油酯的脂质分子。
本发明术语“聚乙二醇(PEG)-脂质缀合物”是指包含脂质部分和聚乙二醇部分的分子。
本发明术语“脂质纳米颗粒”是指具有至少一个纳米量级尺寸的颗粒,其包含至少一种脂质。
本发明术语“疫苗”是指适合于应用于动物(包括人)的组合物,在施用后诱导免疫应答,其强度足以最低限度地帮助预防、改善或治愈起因于由微生物感染的临床疾病。
本发明术语“递送系统”是指调控生物活性成分在空间、时间及剂量在生物体内分布的制剂或组合物。
本发明术语,N/P为阳离子脂质中N与mRNA单核苷酸中P的摩尔比。
本发明术语“烃基”是指相应的烃失去一个氢原子后剩余的基团,在本发明中特别指脂烃基,例如烷基、烯基、炔基,特别是烷基。
本发明涉及一种阳离子脂质,具有如下式I结构:
其中:
L1和L2至少一个为-O-、-O(C=O)O-、-(C=O)NRa-、-NRa(C=O)-或-NRa-,
并且,
L1或L2中的另一个为-O-、-O(C=O)O-、-(C=O)NRa-、-NRa(C=O)-、-NRa-、-O(C=O)-、-(C=O)O-、-C(=O)-、-S(O)x-、-S-S-、-C(=O)S-、-SC(=O)-、-NRaC(=O)NRa-、-OC(=O)NRa-或-NRaC(=O)O-;
G1和G2各自独立地为未取代的C1-C12亚烷基或C1-C12亚烯基;
G3为C1-C24亚烷基、C1-C24亚烯基、C3-C8亚环烷基、C3-C8亚环烯基;
Ra为H或C1-C12烃基;
R1和R2各自独立地为C6-C24烷基或C6-C24烯基;
R3为H、OH、OR4、CN、-C(=O)OR4、-OC(=O)R4或–NR5C(=O)R4;
R4为C1-C12烃基;
R5为H或C1-C6烃基;
x为0、1或2。
具体地,其中的阳离子脂质式I结构中L1和L2各自独立地选自-O-、-O(C=O)O-、-(C=O)NH-、-NH(C=O)-和-NH-。
具体地,其中的阳离子脂质式I结构中,L1和L2均为-O-,或者,L1和L2均为-O(C=O)O-,或者,L1和L2均为-NH-,或者,L1为-NH(C=O)-,L2为-(C=O)NH-。
具体地,其中的阳离子脂质其有以下结构(IA):
其中:
R6在每次出现时独立地为H、OH或C1-C24烃基;
n为1至15的整数。
具体地,其中的阳离子脂质其有以下结构(IB):
其中y和z各自独立地为1至12的整数。
具体地,其中的阳离子脂质结构中n为2至12的整数,优选的,n为2、3、4、5或
6;其中y和z各自独立地为2至10的整数,优选的,为4至9的整数。
具体地,其中的阳离子脂质结构中R1和R2各自独立地具有以下结构:
其中:
R7a和R7b在每次出现时独立地为H或C1-C12烃基;并且a为2至12的整数,优选的,a为8至12的整数;
其中R7a、R7b和a各自被选择为使得R1和R2各自独立地包含6至20个碳原子。
具体地,其中的阳离子脂质结构中至少一次出现的R7a为H,优选的,R7a在每次出现时为H。
具体地,其中的阳离子脂质结构中至少一次出现的R7b为C1-C8烃基;优选的,其中C1-C8烃基为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正己基或正辛基。
具体地,其中的阳离子脂质结构中R1或R2或两者具有以下结构之一:
具体地,其中的阳离子脂质化合物下结构如下:
本发明涉及一种脂质纳米颗粒,包含:(a)阳离子脂质;(b)非-阳离子脂质;(c)聚乙二醇(PEG)-脂质缀合物。优选地,包含:阳离子脂质、中性磷脂、甾族脂质和/或聚乙二醇(PEG)-脂质缀合物。
具体地,其中的聚乙二醇(PEG)-脂质缀合物选自:2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺
(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)或DMG-PEG2000中的一种或多种组合,优选的为DMG-PEG2000。
具体地,其中的中性脂质选自1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)中的一种或多种组合,优选的为DSPC。
具体地,其中的甾族脂质选自燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇以及经多肽修饰后的胆固醇;羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸中的一种或多种组合,优选的为胆固醇。
具体地,其中的阳离子脂质摩尔含量为20~60%、中性磷脂摩尔含量约为5%~25%、甾族脂质摩尔含量约为25%~55%;聚乙二醇(PEG)-脂质缀合物摩尔含量约为0.5%~15%,
具体地,其中阳离子脂质:中性磷脂:甾族脂质:聚乙二醇(PEG)-脂质缀合物摩尔比为30-60:1-20:20-50:0.1-10,优选的,其中阳离子脂质:中性磷脂:甾族脂质:聚乙二醇(PEG)-脂质缀合物摩尔比为47:10:41.5:1.5或44:10:44:2。
具体地,所述疫苗中还包含其他辅料,其中辅料为醋酸钠、氨丁三醇、磷酸二氢钾、氯化钠、磷酸氢二钠、蔗糖中的一种或多种组合。
具体地,其中纳米颗粒的平均粒径为50~200nm或其中纳米颗粒在中性pH下具有净中性电荷或其中纳米颗粒具有小于0.4的多分散性。
本发明涉及一种脂质纳米颗粒mRNA疫苗的制备方法。具体地,将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后与mRNA混合后制得。
具体地,将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液混合后经超滤、稀释、过滤后制得;优选的,将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液按一定流速比混合后经超滤、稀释、过滤后制得;优选的,其中的超滤方式为切向流过滤;更优选的,其中的混合方式可为湍流混合、层流混合或微流体混合。
具体地,稀释液可为乙酸盐缓冲液、柠檬酸盐缓冲液、磷酸盐缓冲液或tris缓冲液。
具体地,其中缓冲液pH为3~6,浓度为6.25~200mM。
具体地,将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后所得的脂质混合溶液与mRNA稀释后的溶液流速比为1~5:1。
具体地,脂质包封mRNA时的N/P为2-10,优选的N/P为3-9,更优选的,N/P为3、4、5、6、7、8、9。
具体地,其中的超滤液选自由以下组成的组:钠盐和三(羟甲基)氨基甲烷(Tris)盐,优选的,超滤液pH为6.5~8.5。
本发明提供一种流感病毒脂质纳米颗粒mRNA疫苗,具体地,给药方式可为口服、肌肉注射、静脉注射或吸入。
具体地,流感病毒脂质纳米颗粒mRNA疫苗的剂型可为口服制剂、液体制剂、冻干粉剂、注射剂或吸入制剂,优选的,为肌肉注射剂、静脉注射剂、干粉吸入剂或雾化吸入剂。
本发明涉及一种流感病毒脂质纳米颗粒mRNA疫苗,包含:(a)编码流感病毒HA、NA、M蛋白和/或N蛋白的mRNA;(b)阳离子脂质;(c)非-阳离子脂质;(d)聚乙二醇(PEG)-脂质缀合物。
具体地,其中的流感病毒脂质纳米颗粒mRNA疫苗,包含:(a)编码流感病毒HA、NA、M蛋白和/或N蛋白的mRNA;(b)上述阳离子脂质;(c)中性磷脂、甾族脂质;(d)聚乙二醇(PEG)-脂质缀合物。
具体地,其中的流感病毒脂质纳米颗粒mRNA疫苗,或被ALC-0315制备的脂质纳米颗粒包裹。
所述流感疫苗编码抗原源自4种季节性流感毒株选自甲型流感病毒H1N1、H3N3、B型流感病毒的山形株(Yamagata)和/或维多利亚株(Victoria)中的一种或多种的。
具体地,其中mRNA编码的氨基酸序列包含SEQ ID NO:1-8一种或多种所示序列。或者与SEQ ID NO:1-8所示序列具有80%或以上同一性的氨基酸序列,优选具有85%、90%、95%、96%、97%、98%、99%以上或100%同一性的氨基酸序列。
甲型H1N1毒株的HA抗原蛋白的氨基酸序列如SEQ ID NO.1所示;
甲型H3N2毒株的HA抗原蛋白的氨基酸序列如SEQ ID NO.2所示;
乙型Victoria系毒株的HA基因编码的氨基酸序列如SEQ ID NO.3所示;
乙型Yamagata系毒株的HA基因编码的氨基酸序列如SEQ ID NO.4所示;
甲型H1N1毒株的NA抗原蛋白的氨基酸序列如SEQ ID NO.5所示;
甲型H3N2毒株的NA基因编码的氨基酸序列如SEQ ID NO.6所示;
乙型Yamagata系毒株的NA基因编码的氨基酸序列如SEQ ID NO.7所示;
乙型Victoria系毒株的NA基因编码的氨基酸序列如SEQ ID NO.8所示。
本发明选择特定的阳离子脂质并与非-阳离子脂质及聚乙二醇(PEG)-脂质组合制备脂质纳米颗粒,实验发现具有良好的体外稳定性以及小鼠免疫反应。
本发明涉及核酸-脂质纳米颗粒疫苗在制备用于预防癌症、病毒感染、细菌感染、真菌感染的疫苗中的应用。
具体地,其中的病毒感染选自流感病毒,其特征在于,其中的流感病毒可选自甲型流感病毒、B型流感病毒;优选的,其中甲型流感病毒可选自由H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、H17和H18组成的组的血凝素(HA)的甲型流感病毒;更优选的,其中甲型流感病毒选自由N1、N2、N3、N4、N5、N6、N7、N8、N9、N10和N11组成的组的神经氨酸酶(NA)的甲型流感病毒;更优
选的,甲型流感病毒选自由H1N1、H1N2、H2N2、H3N1、H3N2、H3N8、H5N1、H5N2、H5N3、H5N8、H5N9、H7N1、H7N2、H7N3、H7N4、H7N7、H7N9、H9N2、H10N8和H10N7组成的组,优选地选自H1N1、H3N2、H5N1和H5N8。
本发明有益效果:
本发明提供系列新型阳离子脂质,采用脂质纳米颗粒作为递送系统,通过构建全新的阳离子脂质具有更优的理化性质,其包封率、稳定性显著优于已上市的阳离子脂质制备的脂质纳米颗粒递送系统,
本发明提供了一种脂质纳米颗粒的流感病毒mRNA疫苗,。利用该LNP载体包载的流感抗原mRNA制成疫苗,通过两次免疫动物(肌肉注射),动物体内产生了高水平的特异性体液和细胞免疫反应,实验证明了利用新型阳离子脂质制备的疫苗具有较高的免疫效力。
图1所示为LNP制剂稳定性-平均粒径数据图;
图2所示为LNP制剂包封率数据图;
图3所示为LNP制剂mRNA完整性数据图;
图4所示为LNP制剂PDI数据图;
图5所示为BALB/c小鼠免疫程序;
图6所示为BALB/c小鼠模型上HAI抗体滴度;
图7所示为BALB/c小鼠模型上ICS法检测特异性分泌TNFα、IFNγ和IL-2的CD4+T细胞频数;
图8所示为BALB/c小鼠模型上ICS法检测特异性分泌TNFα、IFNγ和IL-2的CD8+T细胞频数。
下面将结合本发明的附图,对本发明中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明的部分实施例,而不是全部。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
化合物1的合成
6-溴己基(2-己基癸基)碳酸酯(1a)的合成
将6-溴正己醇(0.91g,5.0mmol)溶于30mL二氯甲烷中,加入4-二甲氨基吡啶(0.90g,7.5mmol),再分批次加入对硝基氯甲酸苯酯(1.20g,6.0mmol),反应室温搅拌3h,在此反应液中加入2-己基癸醇(1.36g,5.6mmol),混合物在室温下搅拌过夜,TLC显示反应完成后,加入20mL二氯甲烷稀释,然后用30mL饱和食盐水洗涤,有机相用无水硫酸钠干燥,过滤并浓缩,柱层析分离得到6-溴己基(2-己基癸基)碳酸酯1a(1.53g,淡黄色油状物),产率68%。
MS m/z(ESI):449.3[M+1]
化合物1的合成
将6-溴己基(2-己基癸基)碳酸酯(1.12g,2.5mmol)溶于四氢呋喃中,加入乙腈,4-氨基-1-丁醇(89.2mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物1(454mg,淡黄色油状物),产率55%。
MS m/z(ESI):826.9[M+1]
1H NMR(300MHz,CDCl3):δ4.13(t,4H,J=6.6Hz),4.05(d,4H,J=5.7Hz),3.56-3.55(m,2H),2.47-2.42(m,6H),1.72-1.67(m,10H),1.53-1.48(m,8H),1.45-1.28(m,52H),0.69(t,12H,J=6.2Hz)
实施例2
化合物2的合成
7-溴庚基十七烷-9-基碳酸酯(2a)的合成
将7-溴庚醇(0.98g,5.0mmol)溶于30mL二氯甲烷中,加入4-二甲氨基吡啶(1.22g,10mmol),再分批次加入对硝基氯甲酸苯酯(1.11g,5.5mmol),反应室温搅拌3h,在此反应液中加入9-羟基十七醇(1.44g,5.6mmol),混合物在室温下搅拌过夜,TLC显示反应完成后,加入20mL二氯甲烷稀释,然后用30mL饱和食盐水洗涤,有机相用无水硫酸钠干燥,过滤并浓缩,柱层析分离得到7-溴庚基十七烷-9-基碳酸酯2a(1.50g,淡黄色油状物),产率65%。
MS m/z(ESI):477.3[M+1]
十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯(2b)的合成
室温条件下,将7-溴庚基十七烷-9-基碳酸酯(2a)(1.38g,3mmol)溶于20mL乙醇中,加入乙醇胺(2.75g,45mmol),升温至50℃,搅拌8h,监控反应进程,原料消耗完全后降温至45℃旋干除去乙醇,用二氯甲烷溶解粗品,用饱和食盐水洗涤三次,有机相用无水硫酸钠干燥,浓缩得到产品十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯2b(1.35g,淡黄色油状物)。
MS m/z(ESI):458.4[M+1]
5-溴戊基十一烷基碳酸酯(2c)的合成
将5-溴戊醇(0.84g,5.0mmol)溶于30mL二氯甲烷中,加入4-二甲氨基吡啶(1.22g,10mmol),再分批次加入对硝基氯甲酸苯酯(1.11g,5.5mmol),反应室温搅拌3h,在此反应液中加入十一醇(0.97g,5.6mmol),混合物在室温下搅拌过夜,TLC显示反应完成后,加入20mL二氯甲烷稀释,然后用30mL饱和食盐水洗涤,有机相用无水硫酸钠干燥,过滤并浓缩,柱层析分离得到5-溴戊基十一烷基碳酸酯2c(1.20g,淡黄色油状物),产率66%。
MS m/z(ESI):365.2[M+1]
化合物2的合成
将十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯(457mg,1.0mmol)溶于四氢呋喃中,加入乙腈,5-溴戊基十一烷基碳酸酯(437mg,1.2mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物2(440mg,淡黄色油状物),产率57%。
MS m/z(ESI):742.8[M+1]
1H NMR(300MHz,CDCl3):δ4.71-4.68(m,1H),4.15-4.10(m,6H),3.53(t,2H,J=5.4Hz),2.94(br,1H),2.58(t,2H,J=5.4Hz),2.45(t,4H,J=5.7Hz),1.75-1.34(m,62H),0.90(t,9H,J=6.3Hz)
实施例3
化合物3的合成
6-溴己基十一烷基碳酸酯(3a)的合成
将6-溴正己醇(0.91g,5.0mmol)溶于30mL二氯甲烷中,加入4-二甲氨基吡啶(0.90g,7.5mmol),再分批次加入对硝基氯甲酸苯酯(1.20g,6.0mmol),反应室温搅拌3h,在此
反应液中加入十一醇(0.97g,5.6mmol),混合物在室温下搅拌过夜,TLC显示反应完成后,加入20mL二氯甲烷稀释,然后用30mL饱和食盐水洗涤,有机相用无水硫酸钠干燥,过滤并浓缩,柱层析分离得到6-溴己基十一烷基碳酸酯3a(1.25g,淡黄色油状物),产率66%。
MS m/z(ESI):379.2[M+1]
化合物3的合成
将6-溴己基十一烷基碳酸酯(948mg,2.5mmol)溶于四氢呋喃中,加入乙腈,4-氨基-1-丁醇(89.2mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物3(412mg,淡黄色油状物),产率60%。
MS m/z(ESI):686.8[M+1]
1H NMR(300MHz,CDCl3):δ4.13(t,8H,J=6.6Hz),3.58(t,2H,J=5.7Hz),2.52(t,6H,J=8.4Hz),1.74-1.64(m,12H),1.63-1.53(m,5H),1.52-1.39(m,39H),0.86(t,6H,J=6.2Hz)
实施例4
化合物4的合成
6-溴己基十七烷-9-基碳酸酯(4a)的合成
将6-溴正己醇(0.91g,5.0mmol)溶于30mL二氯甲烷中,加入4-二甲氨基吡啶(0.90g,7.5mmol),再分批次加入对硝基氯甲酸苯酯(1.20g,6.0mmol),反应室温搅拌3h,在此反应液中加入9-十七醇(1.44g,5.6mmol),混合物在室温下搅拌过夜,TLC显示反应完成后,加入20mL二氯甲烷稀释,然后用30mL饱和食盐水洗涤,有机相用无水硫酸钠干燥,过滤并浓缩,柱层析分离得到6-溴己基十七烷-9-基碳酸酯4a(1.53g,淡黄色油状物),产率66%。
MS m/z(ESI):464.3[M+1]
化合物4的合成
将6-溴己基十七烷-9-基碳酸酯(1.16g,2.5mmol)溶于四氢呋喃中,加入乙腈,4-氨基-1-丁醇(89.2mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),
在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物4(502mg,淡黄色油状物),产率59%。
MS m/z(ESI):855.4[M+1]
1H NMR(300MHz,CDCl3):δ4.71-4.68(m,2H),4.13(t,4H,J=6.6Hz),3.57(t,2H,J=5.4Hz),2.49-2.44(m,6H),1.74-1.28(m,76H),0.90(t,12H,J=6.3Hz)
实施例5
化合物5的合成
将6-溴己基(2-己基癸基)碳酸酯(1.12g,2.5mmol)溶于四氢呋喃中,加入乙腈,乙醇胺(61.0mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物5(487mg,淡黄色油状物),产率61%。
MS m/z(ESI):798.9[M+1]
1H NMR(300MHz,CDCl3):δ4.14(t,4H,J=6.6Hz),4.04(d,4H,J=5.7Hz),3.54(t,2H,J=5.4Hz),2.58(t,2H,J=5.4Hz),2.46(t,4H,J=7.2Hz),1.72-1.65(m,6H),1.49-1.28(m,61H),0.69(t,12H,J=6.2Hz)
实施例6
化合物6的合成
将5-溴戊基十一烷基碳酸酯(910mg,2.5mmol)溶于四氢呋喃中,加入乙腈,乙醇胺(61.0mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物6(410mg,淡黄色油状物),产率65%。
MS m/z(ESI):630.7[M+1]
1H NMR(300MHz,CDCl3):δ4.10(t,8H,J=6.6Hz),3.52(d,2H,J=5.4Hz),2.83(br,1H),2.57(t,2H,J=5.4Hz),2.45(t,4H,J=7.2Hz),1.73-1.62(m,8H),1.52-1.39(m,40H),0.69(t,6H,J=6.2Hz)
实施例7
化合物7的合成
将6-溴己基(2-己基癸基)碳酸酯(1.12g,2.5mmol)溶于四氢呋喃中,加入乙腈,3-甲氧基丙胺(89mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物7(495mg,淡黄色油状物),产率60%。
MS m/z(ESI):826.7[M+1]
实施例8
化合物8的合成
将6-溴己基(2-己基癸基)碳酸酯(1.12g,2.5mmol)溶于四氢呋喃中,加入乙腈,3-氨基丙腈(70mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物8(469mg,淡黄色油状物),产率58%。
MS m/z(ESI):807.7[M+1]
实施例9
化合物9的合成
将6-溴己基(2-己基癸基)碳酸酯(1.12g,2.5mmol)溶于四氢呋喃中,加入乙腈,4-氨基丁酸乙酯盐酸盐(167mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物9(546mg,淡黄色油状物),产率63%。
MS m/z(ESI):868.8[M+1]
实施例10
化合物10的合成
将6-溴己基(2-己基癸基)碳酸酯(1.12g,2.5mmol)溶于四氢呋喃中,加入乙腈,N-(4-氨基丁基)-乙酰胺盐酸盐(167mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物10(560mg,淡黄色油状物),产率69%。
MS m/z(ESI):867.8[M+1]
实施例11
化合物11的合成
8-溴-N-(十七烷-9-基)辛酰胺(11a)的合成
将8-溴辛酸(1.12g,5.0mmol)溶于50mL二氯甲烷中,在0℃下分批加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(1.05g,5.5mmol),搅拌30min后,在反应液中逐滴加入9-氨基十七烷(1.28g,5.0mmol),滴加完毕后,混合物在室温下搅拌过夜,TLC显示反应完成后,用100ml水洗涤2次,有机相用无水硫酸钠干燥,过滤并浓缩,得到化合物11a(1.95g,黄色油状物),产率82%。
MS m/z(ESI):461.3[M+1]。
化合物11b的合成
将8-溴-N-(十七烷-9-基)辛酰胺(1.15g,2.5mmol)溶于四氢呋喃中,加入乙腈,4-
氨基-1-丁醇(89.2mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物11b(534mg,淡黄色油状物),产率63%。
MS m/z(ESI):848.8[M+1];
1H NMR(300MHz,CDCl3):δ8.10(s,2H),4.21(s,1H),3.46-3.4(m,4H),3.02(t,6H,J=6.2Hz),2.14(t,4H,J=4.8Hz),1.57-1.47(t,14H,J=6.3Hz),1.36-1.26(m,66H),0.90(t,12H,J=6.3Hz)。
化合物11的合成
在0℃下,将化合物11b(1.70g,2mmol)缓慢加入四氢铝锂(379mg,10mmol)的无水四氢呋喃(10ml)溶液中,混合物加热回流5小时。反应完全后,降温,在体系中加入水使过量的还原剂完全分解。过滤,滤渣用乙酸乙酯洗涤,得到的滤液用水洗涤,经无水硫酸钠干燥,过滤并浓缩,得到化合物11(1.45g,黄色油状物),产率90%。
MS m/z(ESI):820.8[M+1];
1H NMR(300MHz,CDCl3):δ4.11(s,1H),3.44(t,2H,J=4.8Hz),3.32(s,2H),3.00(t,6H,J=6.3Hz),2.52(t,4H,J=6.3Hz),2.48-2.43(m,2H),1.61-1.56(m,2H),1.36-1.26(m,82H),0.86(t,12H,J=4.8Hz)。
实施例12脂质纳米颗粒包裹流感病毒HA的mRNA抗原
本发明分别使用阳离子脂质1~14制备脂质纳米颗粒核酸疫苗,14种阳离子脂质结构如下表。
表1:阳离子脂质结构式
100mM醋酸钠缓冲液(pH 4.0)稀释流感病毒mRNA疫苗原液至浓度为120μg/ml,该疫苗原液含有编码流感病毒HA蛋白,抗原序列如SEQ ID NO:1所述,目的抗原经过常规修饰,其中N端含有5’UTR及帽子结构,C端含有3’UTR和PolyA尾等设计;稀释后的疫苗原液按照阳离子脂质:DSPC:胆固醇:DMG-PEG2000摩尔比为44:10:44:2配制脂质混合溶液;设定纳米药物制造设备总流速12ml/min、mRNA溶液与脂质混合溶液流速比4:1并开始包封,包封完成后,切向流过滤系统超滤换液收集样品,并加入蔗糖溶液。在N/P(可电离的阳离子脂质与核苷酸磷酸盐)摩尔比不同条件下进行试验(N/P
摩尔比分别为4、6、8)。取样检测包封率、平均粒径、PDI及Zeta电位,结果如下表。
表2:不同阳离子脂质包封后脂质纳米颗粒mRNA疫苗检测结果
由以上结果可以看出,在相同N/P条件下,阳离子脂质1、2、6、7、8、9、10、11、12、13和14所制得样品的包封率均高于阳离子脂质3制得的样品,并且显著高于对照阳离子脂质4和5所制得的样品,初步可以得出阳离子脂质1、2、6、7、8、9、10、11、12、13和14对mRNA抗原具有更好的包封效果。3组的包封率略高于4、5组。
实施例13不同阳离子制得的LNP-mRNA稳定性考察
分别取实施例12中14种不同阳离子脂质制得的LNP-mRNA置于25℃恒温培养箱中分别放置1、2、3、4周考察其稳定性。结果如图1、图2、图3、图4、表3、表4、表5和表6所示。
表3储存稳定性—平均粒径
表4储存稳定性—包封率(%)
表5储存稳定性—mRNA完整性(%)
表6储存稳定性—PDI变化
结果表明,1、2、6、7、8、9、10、11、12、13和14组样品的包封率和mRNA完整性4周内没有明显下降,平均粒径和PDI在4周内没有发生明显增大,并且脂质纳米颗粒的平均粒径和PDI在4周内基本维持不变,与3、4和5组相比,展现出更好的稳定性。3组的稳定性优于4和5组。
实施例14小鼠免疫与检测
1、流感病毒脂质纳米颗粒mRNA疫苗体液免疫评价
将6-8周龄的雌性BALB/c小鼠按8只/组随机分成14组,采用后腿肌肉注射的免疫途径进行免疫。1-14组分别免疫样品1-14(实施例12制备),如图5,分别在第0天和第14天免疫,单次免疫剂量为5μg mRNA-LNP,免疫第14天和28天采血分离血清,进行抗体滴度检测,检测结果如表7和图6所示:
表7不同阳离子脂质包封后脂质纳米颗粒mRNA疫苗抗体滴度
抗体滴度检测结果可以看出,阳离子脂质1、2、6、7、8、9、10、11、12、13和14所制得的脂质纳米颗粒mRNA疫苗能够诱导更高的HAI抗体滴度,3号阳离子脂质效果次之,HAI滴度显著高于对照阳离子脂质4和5所制得的脂质纳米颗粒mRNA疫苗。3组的疫苗滴度高于4和5组。
2、流感病毒脂质纳米颗粒mRNA疫苗细胞免疫反应评价
将实施例制备的样品1-14(编号为mRNA-LNP1~mRNA-LNP14)分别在BALB/c小
鼠模型上进行细胞免疫反应的评价。将6-8周龄的雌性BALB/c小鼠按8只/组随机分成14组,如图5所示,采用后腿肌肉注射的免疫途径进行免疫,在第0天和14天免疫5μg的mRNA-LNP。在第28天,处死小鼠并收获脾细胞,然后使用流感病毒全长HA重叠肽库进行刺激,通过细胞内细胞因子染色流式细胞术(ICS)方法检测细胞因子产生细胞。结果如表8、表9、表10、表11、表12、表13和图7、图8所示。
表8HA特异性CD4+T细胞产生TNF-α的百分比
表9 HA特异性CD4+T细胞产生IFN-γ的百分比
表10 HA特异性CD4+T细胞产生IL-2的百分比
表11 HA特异性CD8+T细胞产生TNF-α的百分比
表12 HA特异性CD8+T细胞产生IFN-γ的百分比
表13 HA特异性CD8+T细胞产生IL-2的百分比
本发明制备的mRNA疫苗不仅诱导了Th1偏向反应,还可以显著激活CD8+T细胞反应,且阳离子脂质1、2、6、7、8、9、10、11、12、13和14所制得的脂质纳米颗粒mRNA疫苗的细胞免疫反应较阳离子脂质3、4和5更优,而阳离子脂质3细胞免疫反应优于4、5阳离子脂质所制得的脂质纳米颗粒mRNA疫苗。
综上,本发明制备的mRNA疫苗显示出较好的用于预防流感病毒的潜力。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
Claims (31)
- 一种阳离子脂质,其特征在于,所述的阳离子脂质具有如下式I结构:
其中:L1和L2至少一个为-O-、-O(C=O)O-、-(C=O)NRa-、-NRa(C=O)-或-NRa-,并且,L1或L2中的另一个为-O-、-O(C=O)O-、-(C=O)NRa-、-NRa(C=O)-、-NRa-、-O(C=O)-、-(C=O)O-、-C(=O)-、-S(O)x-、-S-S-、-C(=O)S-、-SC(=O)-、-NRaC(=O)NRa-、-OC(=O)NRa-或-NRaC(=O)O-;G1和G2各自独立地为未取代的C1-C12亚烷基或C1-C12亚烯基;G3为C1-C24亚烷基、C1-C24亚烯基、C3-C8亚环烷基、C3-C8亚环烯基;Ra为H或C1-C12烃基;R1和R2各自独立地为C6-C24烷基或C6-C24烯基R3为H、OH、OR4、CN、-C(=O)OR4、-OC(=O)R4或–NR5C(=O)R4;R4为C1-C12烃基;R5为H或C1-C6烃基;x为0、1或2。 - 根据权利要求1所述的阳离子脂质,其特征在于,所述的阳离子脂质式I结构中L1和L2各自独立地选自-O-、-O(C=O)O-、-(C=O)NH-、-NH(C=O)-和-NH-。
- 根据权利要求1-2任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质式I结构中所述L1和L2均为-O-,或者,L1和L2均为-O(C=O)O-,或者,L1和L2均为-NH-,或者,L1为-NH(C=O)-,L2为-(C=O)NH-。
- 根据权利要求1-3任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质具有以下结构(IA):
其中:R6在每次出现时独立地为H、OH或C1-C24烃基;n为1至15的整数。 - 根据权利要求1-4任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质其有以下结构(IB):
其中y和z各自独立地为1至12的整数。 - 根据权利要求1-5任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质结构中n为2至12的整数,优选的,n为2、3、4、5或6;其中y和z各自独立地为2至10的整数,优选的,为4至9的整数。
- 根据权利要求1-6任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质结构中R1和R2各自独立地具有以下结构:
其中:R7a和R7b在每次出现时独立地为H或C1-C12烃基;并且a为2至12的整数,优选的,a为8至12的整数;其中R7a、R7b和a各自被选择为使得R1和R2各自独立地包含6至20个碳原子。 - 根据权利要求1-7任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质结构中至少一次出现的R7a为H,优选的,R7a在每次出现时为H。
- 根据权利要求1-8任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质结构中至少一次出现的R7b为C1-C8烃基;优选的,其中C1-C8烃基为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正己基或正辛基。
- 根据权利要求1-9任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质结构中R1或R2或两者具有以下结构之一:
- 根据权利要求1-10任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质化合物下结构如下:
- 一种脂质纳米颗粒,其特征在于,包含:如权利要求1-11任一项所述的阳离子脂质、非-阳离子脂质和/或聚乙二醇(PEG)-脂质缀合物,优选地,包含:阳离子脂质、中性磷脂、甾族脂质和/或聚乙二醇(PEG)-脂质缀合物。
- 根据权利要求12所述的脂质纳米颗粒,其特征在于,所述的聚乙二醇(PEG)-脂质缀合物选自:2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG- 二硬脂基、PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)或DMG-PEG2000中的一种或多种组合,优选的为DMG-PEG2000。
- 根据权利要求12-13任一项所述的脂质纳米颗粒,其特征在于,所述的中性脂质选自1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)中的一种或多种组合,优选的为DSPC。
- 根据权利要求12-14任一项所述的脂质纳米颗粒,其特征在于,所述的甾族脂质选自燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇以及经多肽修饰后的胆固醇;羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸中的一种或多种组合,优选的为胆固醇。
- 根据权利要求12-15任一项所述的脂质纳米颗粒,其特征在于,所述的阳离子脂质在脂质组分中的摩尔百分含量为20~60%、中性磷脂在脂质组分中的摩尔百分含量为5%~25%、甾族脂质在脂质组分中的摩尔百分含量为25%~55%;聚乙二醇(PEG)-脂质缀合物在脂质组分中的摩尔百分含量为0.5%~15%。
- 根据权利要求12-16任一项所述的脂质纳米颗粒,其特征在于,所述阳离子脂质:中性磷脂:甾族脂质:聚乙二醇(PEG)-脂质缀合物摩尔比为30-60:1-20:20-50:0.1-10,优选的,所述阳离子脂质:中性磷脂:甾族脂质:聚乙二醇(PEG)-脂质缀合物摩尔比为47:10:41.5:1.5或44:10:44:2。
- 根据权利要求12-17任一项所述的脂质纳米颗粒,其特征在于,所述疫苗中还包含其 他辅料,所述辅料为醋酸钠、氨丁三醇、磷酸二氢钾、氯化钠、磷酸氢二钠、蔗糖中的一种或多种的组合。
- 根据权利要求12-18任一项所述的脂质纳米颗粒,其特征在于,所述纳米颗粒的平均粒径为50~200nm或所述纳米颗粒在中性pH下具有净中性电荷或所述纳米颗粒具有小于0.4的多分散性。
- 一种根据权利要求12-19任一项所述的脂质纳米颗粒的制备方法,其特征在于,包括将所述阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后与mRNA混合的步骤。
- 根据权利要求20所述的脂质纳米颗粒的制备方法,其特征在于,将所述阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液混合后经超滤、稀释、过滤后制得;优选的,将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液按一定流速比混合后经超滤、稀释、过滤后制得;优选的,所述的超滤方式为切向流过滤;更优选的,所述的混合方式可为湍流混合、层流混合或微流体混合。
- 根据权利要求21所述的脂质纳米颗粒的制备方法,其特征在于,稀释液可为乙酸盐缓冲液、柠檬酸盐缓冲液、磷酸盐缓冲液或tris缓冲液。
- 根据权利要求22所述的脂质纳米颗粒的制备方法,其特征在于,所述缓冲液pH为3~6,浓度为6.25~200mM。
- 根据权利要求21-23任一项所述的脂质纳米颗粒的制备方法,其特征在于,将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后所得的脂质混合溶液与mRNA稀释后的溶液流速比为1~5:1。
- 根据权利要求21-24任一项所述的脂质纳米颗粒的制备方法,其特征在于,脂质包封mRNA时的N/P为2-10,优选的N/P为3-9,更优选的,N/P为3、4、5、6、7、8、9,所述的N/P为阳离子脂质中N与mRNA单核苷酸中P的摩尔比。
- 根据权利要求21-25任一项所述的脂质纳米颗粒的制备方法,其特征在于,所述的超滤液选自由以下组成的组:钠盐和三(羟甲基)氨基甲烷(Tris)盐,优选的,超滤液pH为6.5~8.5。
- 根据权利要求21-26任一项所述的脂质纳米颗粒的制备方法,其特征在于,其剂型为口服制剂、液体制剂、冻干粉剂、注射剂或吸入制剂,优选的,为肌肉注射剂、静脉注射剂、干粉吸入剂或雾化吸入剂。
- 一种流感病毒脂质纳米颗粒mRNA疫苗,其特征在于,包含:编码流感病毒血凝素蛋白HA、NA、M蛋白和/或N蛋白的mRNA,所述mRNA被权利要求12-27任一项所述的脂质纳米颗粒包裹或被ALC-0315制备的脂质纳米颗粒包裹。
- 根据权利要求28所述的流感病毒脂质纳米颗粒mRNA疫苗,所述流感疫苗编码抗原源自4种季节性流感毒株选自甲型流感病毒H1N1、H3N3、B型流感病毒的山形株(Yamagata)和/或维多利亚株(Victoria)中的一种或多种的。
- 一种权利要求28-29任一项所述的流感病毒脂质纳米颗粒mRNA疫苗在制备用于预防流感的药物中的用途。
- 根据权利要求30所述的用途,其特征在于,所述的流感病毒选自甲型流感病毒、B型流感病毒;优选的,所述甲型流感病毒选自由H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、H17和H18组成的组的血凝素(HA)的甲型流感病毒;更优选的,所述甲型流感病毒选自由N1、N2、N3、N4、N5、N6、N7、N8、N9、N10和N11组成的组的神经氨酸酶(NA)的甲型流感病毒;更优选的,甲型流感病毒选自由H1N1、H1N2、H2N2、H3N1、H3N2、H3N8、H5N1、H5N2、H5N3、H5N8、H5N9、H7N1、H7N2、H7N3、H7N4、H7N7、H7N9、H9N2、H10N8和H10N7组成的组,优选地选自H1N1、H3N2、H5N1和H5N8。
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