CN114277039A - 呼吸道合胞病毒mRNA疫苗及其制备方法和应用 - Google Patents
呼吸道合胞病毒mRNA疫苗及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及核酸疫苗领域，尤其涉及呼吸道合胞病毒mRNA疫苗及其制备方法和应用。本发明提供的编码呼吸道合胞病毒抗原的mRNA含有编码呼吸道合胞病毒的F蛋白的编码区，该mRNA递送至体内可以诱导中和抗体反应及CD4+和CD8+T细胞应答，且表达RSV F的mRNA疫苗不会导致实验动物产生疫苗增强型呼吸道疾病。
Description
技术领域
本发明涉及核酸疫苗领域，尤其涉及呼吸道合胞病毒mRNA疫苗及其制备方法和应用。
背景技术
呼吸道合胞病毒(RSV)是引起上、下呼吸道疾病的肺病毒科成员之一，在婴儿、免疫力低下者和老年人中发病率很高。在世界范围内，约1.5％的婴儿因呼吸道合胞病毒下呼吸道感染(LRTI)住院。在成人中，呼吸道合胞病毒感染的严重程度随年龄增长而增加。尽管RSV疫苗是有效的预防途径，但目前尚未有RSV疫苗得获许可上市。
RSV F蛋白是一种Ⅰ型融合糖蛋白，在各型别之间相对保守，是一个理想的疫苗靶点，天然RSV感染引起的中和抗体主要针对F蛋白。F蛋白在两个构象之间的转换：亚稳态的融合前构象和稳定的融合后构象。尽管中和抗体靶向的抗原表位存在于F蛋白两种构象上，但人类对RSV感染的自然免疫应答特征表明，大多数RSV中和抗体结合F蛋白的融合前构象。然而F蛋白的融合前构象具有不稳定性，因此，基于RSV灭活疫苗或直接从RSV中提取的F蛋白亚单位疫苗均以失败告终。
开发RSV疫苗对婴幼儿和老年目标人群提出了不同的挑战。在未感染过RSV的婴儿中，通过被动接受靶向RSV-F的单克隆抗体Palivizumab，可以预防RSV引起的严重LRTI。然而，采用福尔马林灭活RSV候选疫苗(FI-RSV)或F蛋白亚单位疫苗却能够引起的免疫病理反应，即疫苗增强型呼吸道疾病(VERD)。与婴儿相比，绝大多数成人已自然感染RSV，并具有可检测的中和抗体滴度。老年人由于对呼吸道合胞病毒F和G蛋白的体液反应较低，也是呼吸道合胞病毒病感染的高危人群。细胞免疫减弱也可能在老年人呼吸道合胞病毒感染中发挥作用，随着年龄的增长，RSV特异性CD8+T细胞减少，调节性T细胞数量增加，且倾向于Th2功能表型，而Th2功能表型的倾向性与疫苗增强型呼吸道疾病的发生密切相关。
由脂质纳米粒(LNPs)包裹的体外转录的mRNAs组成的核酸疫苗可以引发强烈的体液和细胞免疫反应。通过LNP递送mRNA抗原可提高对疫苗的免疫应答，LNP可以保护mRNA不受酶降解，并促进靶细胞对mRNA的有效摄取和细胞内释放。但目前尚未见有效防治RSV的mRNA疫苗的相关报道。
发明内容
有鉴于此，本发明要解决的技术问题在于提供有效防治RSV感染的呼吸道合胞病毒mRNA疫苗及其制备方法和应用。
本发明提供了编码呼吸道合胞病毒F蛋白的核酸，包括I)～IV)中至少一种：
I)、具有如SEQ ID NO:1或SEQ ID NO:2所示核苷酸序列的核酸；
II)、在I)所述的片段中取代、缺失或添加一个或多个核苷酸的核酸；
III)、与如I所示的核苷酸序列具有至少70％同源性的序列且编码呼吸道合胞病毒F蛋白的核酸；
IV)、与I)～III)中任一项部分互补或完全互补的核酸。
具体实施例中，本发明提供的编码呼吸道合胞病毒F蛋白的核酸的序列如SEQ IDNO:1或SEQ ID NO:2所示。其中，SEQ ID NO:1所述序列的核酸编码RSV A型的F蛋白，SEQ IDNO:2所述序列的核酸编码RSV B型的F蛋白。
本发明还提供了核糖核酸片段，其包括本发明所述的核酸，还包括5’-帽结构、5’-UTR、3’-UTR和3’端PolyA中至少一种。
本发明所述的核糖核酸片段中，所述：
5’-帽结构选自dmCAP、mCAP、tmCAP或ARCA中至少一种；
5’-UTR选自X-GLOBIN或A-GLOBIN；
3’-UTR选自X-GLOBIN、A-GLOBIN或脂氧合酶基因；
3’端PolyA的长度为20～100。
所述核糖核酸片段由5’端至3’端依次包括：
ARCA、X-GLOBIN 5’-UTR、SEQ ID NO:1所示序列的核酸、X-GLOBIN3’-UTR和长度为50的PolyA；
或包括ARCA、A-GLOBIN 5’-UTR、SEQ ID NO:1所示序列的核酸、A-GLOBIN 3’-UTR和长度为40的PolyA；
或包括ARCA、X-GLOBIN 5’-UTR、SEQ ID NO:1所示序列的核酸、脂氧合酶基因3’-UTR和长度为30的PolyA；
或包括ARCA、A-GLOBIN 5’-UTR、SEQ ID NO:1所示序列的核酸、脂氧合酶基因3’-UTR和长度为70的PolyA；
或包括ARCA、X-GLOBIN 5’-UTR、SEQ ID NO:2所示序列的核酸、X-GLOBIN 3’-UTR和长度为80的PolyA；
或包括ARCA、A-GLOBIN 5’-UTR、SEQ ID NO:2所示序列的核酸、A-GLOBIN 3’-UTR和长度为50的PolyA。
本发明所述的核糖核酸片段中，
ARCA的结构为m7G(5')ppp(5')(2'-OMeA)pG。
X-GLOBIN 5’-UTR的核酸序列如SEQ ID NO:3所示；
A-GLOBIN 5’-UTR的核酸序列如SEQ ID NO:4所示；
X-GLOBIN 3’-UTR的核酸序列如SEQ ID NO:5所示；
A-GLOBIN 3’-UTR的核酸序列如SEQ ID NO:6所示；
脂氧合酶基因3’-UTR的核酸序列如SEQ ID NO:7所示。
本发明所述的核酸或所述的核糖核酸片段在制备防治RSV病毒感染的疫苗中的应用。
本发明还提供了一种疫苗，其包括所述的核糖核酸片段、疫苗载体和辅料。
所述疫苗载体由可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂制得。一些实施例中，所述疫苗载体中，各组分的摩尔比为可质子化阳离子脂质：结构脂质：辅助脂质：表面活性剂＝(30～55)：(20～45)：(5～15)：(1～5)。
所述可质子化阳离子脂质选自DOTAP、DODMA、Dlin-MC3-DMA和DlinDMA中的至少一种。
所述结构脂质选自胆固醇或胆固醇衍生物中的至少一种。
所述辅助脂质选自DSPC、DOPC、DOPE和DOPS中的至少一种。
所述表面活性剂选自PEG-DMG和PEG-DSPE中的至少一种。
所述辅料为盐或盐的水溶液；所述盐选自柠檬酸盐、乙酸盐或磷酸盐中至少一种。
所述疫苗制备过程中，水相的pH值为3.0～7.0，其中所述的水相中核糖核酸片段的含量为0.01mg/mL～2.0mg/mL。
所述疫苗载体与所述的核糖核酸片段的质量比为(1～60):1。一些实施例中，所述疫苗载体与所述的核糖核酸片段的质量比为(20～50):1，一个具体实施例中，所述疫苗载体与所述的核糖核酸片段的质量比为40:1。
本发明所述疫苗的剂型为注射剂或鼻腔喷雾剂。
所述疫苗的制备方法其包括：
将所述的核糖核酸片段溶于辅料水溶液，得到水相组分；
将可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂溶于有机溶液得到有机相组分；所述有机溶剂为无水乙醇、异丙醇或丙酮；
将水相组分和有机相组分混合得到疫苗；或者以辅料水溶液稀释有机相组分后经浓缩得到疫苗。
本发明所述疫苗的制备方法中，水相组分pH值为3.0～7.0，其中所述的核糖核酸片段在水相中的含量为0.01mg/mL～2.0mg/mL；所述疫苗载体与核糖核酸片段的质量比为(1～60):1。本发明所述的疫苗制备方法中，有机相中可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂的总浓度为5～10mg/mL。
本发明实施例中，水相中mRNA的浓度为0.01～2.0mg/mL。
本发明实施例中，有机相和水相的体积比为1:(1～10)。
一些实施例中，所述混匀采用微流控设备，流速范围为6～24mL/min，优选为12.0mL/min。
另一些实施例中，替换混合液中的溶媒为pH值为7.0～8.0的辅料水溶液包括：用辅料水溶液将混合液稀释50～100倍，再浓缩。
本发明还提供了一种防治呼吸道合胞病毒感染的方法，其为给予本发明所述的疫苗。
本发明所述疫苗的给药方式包括肌肉注射、鼻腔喷雾，优选为肌肉注射。
与现有技术相比，本发明的有益效果为：
本发明提供一种优化RSV A型和B型F蛋白mRNA，转录其的核苷酸序列如SEQ IDNo.1和SEQ ID No.2所示。发明人对呼吸道合胞病毒A2和B型毒株F蛋白(RSV)编码区进行优化，序列优化包括：针对在人体或小鼠内表达时的密码子偏好性进行调整、常用密码子的使用频率进行调整，得到核苷酸序列如SEQ ID No.1和SEQ ID No.2所示的优化RSV F蛋白mRNA的序列。该核苷酸序列使得转录后的mRNA结构更稳定，在哺乳动物和人体内的目标蛋白翻译效率更高。
本发明提供的呼吸道合胞病毒核酸疫苗，包括优化RSV A型和/或B型F蛋白mRNA和疫苗载体。该核酸疫苗将体外转录合成的优化RSV F蛋白mRNA，递送至体内产生呼吸道合胞病毒F糖蛋白，实现在细胞表面表达天然结构RSV F蛋白的效果，该方法能够有效产生天然构象的RSV F蛋白，诱导产生具有中和活性的特异性抗体及Th1型免疫应答，避免传统疫苗产生的疫苗增强型呼吸道疾病(VERD)。
附图说明
图1示实施例1中不同非编码区序列及Poly A尾长度对蛋白表达量的影响结果；
图2示实施例5中棉鼠试验呼吸道合胞病毒核酸疫苗的中和抗体滴度结果，其纵坐标为呼吸道合胞病毒中和抗体滴度，其中control组为给予生理盐水的水平。
具体实施方式
本发明提供了呼吸道合胞病毒mRNA疫苗及其制备方法和应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
除非另有说明，本文中所用的专业与科学术语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法或材料也可应用于本发明中。
本发明提供的编码呼吸道合胞病毒F蛋白的核酸中，包括编码RSV病毒A型F蛋白和RSV病毒B型F蛋白的核酸。所述核酸为mRNA，用于制备mRNA疫苗。本发明提供的mRNA对编码区继续进行了优化，序列优化包括：针对在小鼠及人体内表达时的密码子偏好性进行调整、常用密码子的使用频率进行调整，得到核苷酸序列如SEQ ID No.1和SEQ ID No.2所示。
其中，SEQ ID No.1所示序列的核酸编码RSV A型的F蛋白，具体序列为：
5’-auggagcugcccauccugaacaccaacgccaucaccaccauucucgccgccgugacccugugcuucgcuagcagccaaaacaucaccgaggaguucuaucagagcaccugcagcgccgucagcaagggcuaccugagcgcccugagaaccggcugguacacaagcgugaucaccaucgagcugagcaacaucaaggagaacaagugcaacggcaccgacgccaaggugaagcugaucaagcaagagcuggacaaguacaagaacgccgugaccgagcugcagcugcugaugcagagcacccccgccgccaacagcagagcuagaagagagcugccuagauucaugaacuacacccugaacaacaccaagaacaccaacgugacccugagcaagaaaagaaagagaagauuccugggcuuccugcugggcgugggcagcgccaucgcuagcggcaucgccgugagcaaagugcugcaccucgagggcgaggugaacaagaucaagagcgcccugcugagcaccaacaaggccguggugagccugagcaacggcgugagcgugcugacaagcaaggugcuggaucucaagaacuacaucgacaagcagcugcugcccaucgugaauaaacagagcuguagcaucagcaacaucgagaccgugaucgaguuucagcagaagaacaacagacugcuggagaucacaagagaguucagcgucaacgcuggcgugaccacccccgugagcaccuacaugcugaccaacagcgagcugcugagccugaucaacgacaugcccaucaccaacgaucagaagaagcugaugagcaacaacgugcagaucgugagacagcagagcuacagcaucaugagcaucaucaaggaagaggugcuggccuacguggugcagcugccccuguacggcgugaucgacacccccugcuggaagcugcacacaagcccccugugcaccacaaacacaaaagagggcagcaacaucugccugacaagaaccgacagaggcugguacugcgacaacgccggcagcgugagcuucuucccccaagccgagaccugcaaggugcagagcaacagaguguucugcgacaccaugaacagccugacccugccuagcgaggugaaccugugcaacaucgacaucuucaaccccaaguacgacugcaagaucaugacaagcaagaccgacgugagcagcuccgugauuacaagccugggcgccaucgugagcugcuacggcaagaccaagugcaccgcuagcaacaagaacagaggcaucaucaagaccuucagcaacggcugcgacuacgugagcaacaagggcguggacaccgugagcgugggcaacacccuguacuacgugaacaagcaagagggcaagagccuguacgugaagggcgagcccaucaucaacuucuacgacccccugguguucccuagcgacgaguucgacgcuagcaucagccaagugaacgagaagaucaaucagagccuggccuucaucagaaagagcgacgagcugcugcacaacgugaacgccggcaagagcaccaccaauauuaugaucaccaccaucauuaucgugaucauugugauccugcucagccugaucgccgugggccugcuccuguacugcaaggcuagaagcacccccgugacacugagcaaggaucagcugagcggcaucaacaacaucgccuucagcaacuga-3’；
其中，SEQ ID No.2所示序列的核酸编码RSV B型的F蛋白，具体序列为：
5’-auggagcugcugauccauagauccagugcgauuuuucugacccuggcuaucaaugcccucuaucucacuucaucacagaauauuacagaagaguuuuaucaaaguaccugcuccgccguguccagaggcuauuuguccgcacuuaggacuggaugguauacuuccgugauuacuauagagcuguccaauauaaaggagacaaagugcaauggcaccgacacaaaagugaaacugauuaagcaagaguuggacaaauauaaaaaugcaguaaccgagcugcagcugcuuaugcagaacaccccagccgucaauaacagggcucgaagggaagccccucaguauaugaacuacacuauaaacaccacaaagaaucucaacguuuccaucuccaaaaaacgcaaacggcgcuucuugggauuucugcugggcguugguagugccauagccuccggaauagcaguaagcaaggugcugcaccucgagggggaagugaacaagaucaagaacgcccugcagcugacuaacaaagccgucgucagccugucuaaugguguuucaguccugacuuccaagguucucgacuugaaaaauuacauuaacaaucagcugcucccuaucgugaaucagcagucaugcaggaucuccaauaucgagaccgugauugaguuucagcaaaagaauagcaggcucuuggaaaucacuagagaguuuuccguuaaugccggagugaccacuccccuguccacuuacaugcugacaaacagugaacuguugucccugaucaaugauaugccaauuacaaaugaccaaaaaaaacuuaugucuucaaaugugcagaucgugcggcaacaguccuacagcauuaugagcaucaucaaagaggagguguuggccuacguggugcaguugcccauuuacggcgugaucgacacucccugcuggaagcuccauaccuccccacugugcacaaccaauaccaaagaaggguccaacaucugccuuacacggacggaucgcgggugguacugugauaaugcaggcucuguaucuuuuuucccgcaggccgauacaugcaaggugcaaagcaauagaguuuucugugauacgaugaacagccuuacgcucccgagugaggucucacucuguaacaccgacaucuuuaacagcaaauaugauugcaaaaucaugaccagcaagacagacauuucaagcuccgugaucacuagccucggugccauaguuagcugcuacggcaagacaaagugcaccgccucuaacaagaaccgcggcauuaucaaaacauuuucuaauggcugcgauuaugugucuaauaaaggcguugacacgguuuccgucgggaauacucuguauuaugugaauaagcuggagggaaagaacuuguauguuaagggggaaccuaucaucaacuacuacgacccguugguuuuuccgagcgacgaguucgacgccuccaucagccaggugaacgagaagaucaaucagucacuugcguuuauacgaagguccgaugagcuccugcacaaugucaacacugguaaaucaacaaccaacaucaugaucacagccaucaucaucguuaucauagugguacuccugucucugaucgcuaucgggcugcugcuguacuguaaagccaagaacaccccagugacucugucuaaggaccagcugagcggcaucaauaacauugcuuucagcaaauag-3’
相对于野生型序列而言，本发明提供的优化后的SEQ ID NO：1～2所示的核苷酸片段使得转录后的mRNA结构更稳定，在小鼠和人体内的目标蛋白翻译效率更高。
进一步的，在优化后核酸的5’端和3’端添加提高序列稳定性，提高蛋白质翻译效率的元件，获得本发明所述的核糖核酸片段。所述元件包括5’-帽结构、5’-UTR、3’-UTR和3’端PolyA中至少一种。
在优选地实施方式中，5’-UTR的长度为10～100个核苷酸，更优选为20-80个核苷酸。
在优选地实施方式中，5’-UTR为X-GLOBIN 5’-UTR序列(SEQ ID No.3)、A-GLOBIN5’-UTR序列(SEQ ID No.4)。
在优选地实施方式中，3’-UTR包括珠蛋白X-GLOBIN 3’-UTR序列(SEQ ID No.5)、Α-GLOBIN 3’-UTR(SEQ ID No.6)序列或脂氧合酶基因3’-UTR序列(SEQ ID No.7)。
一些具体实施例中，所述核糖核酸片段由5’端至3’端依次包括：
ARCA、X-GLOBIN 5’-UTR、SEQ ID NO:1所示序列的核酸、X-GLOBIN3’-UTR和长度为50的PolyA。该片段在本发明中记为RSV F-1，其具体包括5’端至3’端顺序连接的SEQ IDNO:3所示序列的片段、SEQ ID NO:1所示序列的片段、SEQ ID NO:5所示序列的片段和长度为50的PolyA。
或包括ARCA、A-GLOBIN 5’-UTR、SEQ ID NO:1所示序列的核酸、A-GLOBIN 3’-UTR和长度为40的PolyA。该片段在本发明中记为RSV F-2，其具体包括5’端至3’端顺序连接的SEQ ID NO:4所示序列的片段、SEQ ID NO:1所示序列的片段、SEQ ID NO:6所示序列的片段和长度为40的PolyA。
或包括ARCA、X-GLOBIN 5’-UTR、SEQ ID NO:1所示序列的核酸、脂氧合酶基因3’-UTR和长度为30的PolyA。该片段在本发明中记为RSV F-3，其具体包括5’端至3’端顺序连接的SEQ ID NO:3所示序列的片段、SEQ ID NO:1所示序列的片段、SEQ ID NO:7所示序列的片段和长度为30的PolyA。
或包括ARCA、A-GLOBIN 5’-UTR、SEQ ID NO:1所示序列的核酸、脂氧合酶基因3’-UTR和长度为70的PolyA。该片段在本发明中记为RSV F-4，其具体包括5’端至3’端顺序连接的SEQ ID NO:4所示序列的片段、SEQ ID NO:1所示序列的片段、SEQ ID NO:5所示序列的片段和长度为70的PolyA。
或包括ARCA、X-GLOBIN 5’-UTR、SEQ ID NO:2所示序列的核酸、X-GLOBIN 3’-UTR和长度为80的PolyA。该片段在本发明中记为RSV F-5，其具体包括5’端至3’端顺序连接的SEQ ID NO:3所示序列的片段、SEQ ID NO:2所示序列的片段、SEQ ID NO:6所示序列的片段和长度为80的PolyA。
或包括ARCA、A-GLOBIN 5’-UTR、SEQ ID NO:2所示序列的核酸、A-GLOBIN 3’-UTR和长度为50的PolyA。该片段在本发明中记为RSV F-6，其具体包括5’端至3’端顺序连接的SEQ ID NO:4所示序列的片段、SEQ ID NO:2所示序列的片段、SEQ ID NO:7所示序列的片段和长度为50的PolyA。
本发明所述的核酸或所述的核糖核酸片段能够用于制备防治RSV病毒感染的疫苗。该呼吸道合胞病毒核酸疫苗将体外转录合成的优化RSV F蛋白mRNA，递送至体内产生呼吸道合胞病毒融合蛋白F，避免传统疫苗产生的疫苗增强型呼吸道疾病(VERD)。
本发明提供的疫苗，其包括所述的核糖核酸片段、疫苗载体和辅料。
本发明中，疫苗载体为阳离子脂质纳米颗粒。
本发明实施例中，所述阳离子脂质纳米颗粒与优化RSV F蛋白mRNA的质量比为(10～60):1。一些实施例中，所述阳离子脂质纳米颗粒与优化RSV F蛋白mRNA的质量比为为(20～50):1，一些具体实施例中，所述阳离子脂质纳米颗粒与优化RSV F蛋白mRNA的质量比10:1、20:1或40:1。
本发明中，所述阳离子脂质纳米颗粒包括可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂。其中：
所述可质子化阳离子脂质选自DOTAP、DODMA、Dlin-MC3-DMA、DlinDMA中的至少一种。一些实施例中，可质子化阳离子脂质所述DOTAP或Dlin-MC3-DMA。
所述结构脂质选自胆固醇、胆固醇酯或胆汁酸中至少一种，一些实施例中，所述结构脂质为胆固醇。
所述辅助脂质选自二硬脂酰基磷脂酰胆碱(DSPC)、二油酰基卵磷脂(DOPC)、二油酰基磷脂酰乙醇胺(DOPE)或二油酰基磷脂酰丝氨酸(DOPS)中的至少一种，一些实施例中，所述辅助脂质为DSPC。
所述表面活性剂选自PEG-DMG或PEG-DSPE，一些实施例中，所述表面活性剂为PEG-DMG。其中，所述PEG-DMG是1,2-二肉豆蔻酸甘油酯的聚乙二醇(PEG)衍生物，PEG长度为2000；所述PEG-DSPE是二硬脂酰基磷脂酰乙醇胺的聚乙二醇(PEG)衍生物，PEG长度为2000。
一些实施例中，阳离子脂质纳米颗粒包括如下摩尔百分数的30％～55％可质子化阳离子脂质、20％～45％结构脂质、5％～15％辅助脂质和1％～5％表面活性剂。一些具体实施例中，所述阳离子脂质纳米颗粒中可质子化阳离子脂质的摩尔百分比为30％、35％、40％、45％、50％或55％。一些具体实施例中，所述结构脂质的摩尔百分比为20％、25％、30％、35％、40％或45％；一些具体实施例中，所述辅助脂质的摩尔百分比为5％、10％或15％；一些具体实施例中，所述表面活性剂的摩尔百分比为1％、2％、3％、4％或5％。本发明中，可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂的摩尔含量合计100％。
本发明还提供了所述呼吸道合胞病毒核酸疫苗的制备方法，其将本发明提供的优化RSV F蛋白mRNA和疫苗载体混合，得到呼吸道合胞病毒核酸疫苗。
一些实施例中，所述呼吸道合胞病毒核酸疫苗的制备方法包括：
(a)将优化RSV F蛋白mRNA溶于辅料水溶液中，得到水相；所述辅料的水溶液包括但不限于柠檬酸盐缓冲液、乙酸钠溶液或磷酸盐缓冲液，优选为乙酸钠溶液；
(b)按配方比例将可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂溶于有机溶液，得到有机相；所述有机溶液包括无水乙醇、异丙醇或丙酮，优选为无水乙醇；
(c)将(a)得到的水相和(b)得到的有机相混匀得到混合液，替换混合液中的溶媒为缓冲液，得到呼吸道合胞病毒核酸疫苗；优选地，替换的辅料水溶液为磷酸盐缓冲液，pH值7～8。
本发明实施例中，有机相中可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂的总浓度为5～10mg/mL。
本发明实施例中，水相中优化RSV mRNA的浓度为0.01～2.0mg/mL。水相的pH值为3.0～7.0。
本发明实施例中，有机相和水相的体积比为1:1～1:10。
本发明实施例中，混匀采用微流控设备，流速范围为6～24mL/min，优选为12.0mL/min。
本发明实施例中，替换混合液中的溶媒为pH值为7.0～8.0的PBS缓冲液，用缓冲液将混合液稀释50～100倍，再浓缩。替换混合液中的溶媒可以采用切向流过滤或透析方法等。
本发明采用的试材皆为普通市售品，皆可于市场购得。下面结合实施例，进一步阐述本发明：
实施例1RSV F蛋白mRNA的序列优化
本实施例中的RSV F蛋白mRNA的序列优化方案如表1所示，具体如下：
RSV F-1蛋白mRNA序列关键元件是采用X-GLOBIN 5’-UTR、呼吸道合胞病毒08-046972株的F蛋白(RSV A型F蛋白)的开放阅读框(ORF)部分经人源或小鼠源密码子偏好性优化的核苷酸序列(SEQ ID No.1)、X-GLOBIN 3’-UTR和polyA序列以及后续元件。
RSV F-2～RSV F-4蛋白mRNA序列关键元件是与RSV F-1蛋白mRNA的编码区域一致，只是5’-UTR、3’-UTR有所改变；RSV F-5～RSV F-6蛋白mRNA序列进一步地修改了ORF部分序列为RSV B型F蛋白(SEQ ID No.2)部分，如表1所示。
RSV F-1～RSV F-4的ORF部分最终翻译出来的蛋白序列一致，为呼吸道合胞病毒08-046972株F蛋白(GenBank:AFM55552.1)，具体为SEQ ID No.8所示。
RSV F-5～RSV F-6的ORF部分最终翻译出来的蛋白序列一致，为呼吸道合胞病毒B型(GenBank:QKY59503.1株)F蛋白，具体为SEQ ID No.9所示。
表1.候选RSV F蛋白mRNA序列特征
| 序列名称 | 5’-UTR | ORF | 3’UTR | Poly A |
| RSV F-1 | X-GLOBIN 5’-UTR | SEQ ID No.1 | X-GLOBIN 3’-UTR | 50A |
| RSV F-2 | A-GLOBIN 5-UTR | SEQ ID No.1 | A-GLOBIN 3’-UTR | 40A |
| RSV F-3 | X-GLOBIN 5’-UTR | SEQ ID No.1 | 脂氧合酶基因3’-UTR | 30A |
| RSV F-4 | A-GLOBIN 5’-UTR | SEQ ID No.1 | 脂氧合酶基因3’-UTR | 70A |
| RSV F-5 | X-GLOBIN 5’-UTR | SEQ ID No.2 | X-GLOBIN 3’-UTR | 80A |
| RSV F-6 | A-GLOBIN 5-UTR | SEQ ID No.2 | A-GLOBIN 3’-UTR | 50A |
各序列的5’帽子结构采用ARCA，结构为(m7G(5')ppp(5')(2'-OMeA)pG)。体外转录工艺制备出来的上述RSV F-1mRNA、RSV F-2mRNA、RSV F-3mRNA、RSV F-4mRNA、RSV F-5mRNA、RSV F-6mRNA分别转染HEK293细胞。
首先采用4×105个细胞/mL的密度将HEK293细胞加入6孔板中，至细胞融合约为80％时进行细胞转染。将2μg mRNA和转染试剂Lipofectamine 2000(Thermo Fisherscientific)混合后转染HEK293细胞，具体转染操作按照转染试剂产品说明书进行。
转染24小时后的HEK293细胞用抗呼吸道合胞病毒融合蛋白F抗体进行免疫标记，然后使用流式细胞仪检测F蛋白表达量(图1)。荧光素mRNA(Luc mRNA)转染细胞作为阴性对照。
从图中结果可以看出，RSV F-1mRNA转染细胞中的F蛋白表达量高于RSV F-3转染组，可见在元件不变的情况下，3’-UTR含有50个A比含有30个A转染细胞中的F蛋白表达量高；RSV F-4mRNA转染细胞中的F蛋白表达量高于RSV F-2转染组，说明含有70个A比含有40个A转染细胞中的F蛋白表达量高；RSV F-1mRNA转染细胞中的F蛋白表达量高于RSV F-4转染组，RSV F-5mRNA转染细胞中的F蛋白表达量高于RSV F-6转染组，说明X-GLOBIN非编码序列优于A-GLOBIN非编码序列。
鉴于实验表明，RSV F-1和RSV F-5抗原表达量高于其余几组。通过流式细胞结果，RSV F-1和RSV F-5荧光阳性细胞百分含量最高，因此，采用RSV F-1和RSV F-5制备疫苗。
实施例2呼吸道合胞病毒核酸疫苗的制备方法
将实施例1中RSV F-1和RSV F-5的mRNA分别溶解于pH 4的柠檬酸盐缓冲液，将浓度调整为0.05mg/mL，将溶解后的两种mRNA等体积混合，得到水相1。
将实施例1中RSV F-1和RSV F-5的mRNA分别溶解于pH 4的乙酸钠盐缓冲液，并将浓度调整为0.05mg/mL，将溶解后的两种mRNA等体积混合，得到水相2。
将实施例1中RSV F-1和RSV F-5的mRNA分别溶解于pH 4的磷酸盐缓冲液，并将浓度调整为0.05mg/mL，将溶解后的两种mRNA等体积混合，得到水相3。
将配方比例DOTAP、胆固醇、DSPC和PEG-DMG溶于无水乙醇，并将总脂质浓度调整至8mg/mL，得到有机相1。
将配方比例Dlin-MC3-DMA、胆固醇、DSPC和PEG-DMG溶于异丙醇，并将总脂质浓度调整至10mg/mL，得到有机相2。
利用微流控设备混合的方式将有机相与水相按照表2中的组合方式，以6:1的体积比混合。利用微流控设备混合时流速12.0mL/min。
表2候选疫苗制备时的水相和有机相的比例
| 编号 | 水相 | 有机相 | 混合比例 |
| 1 | 水相1 | 有机相1 | 水相:有机相＝6:1 |
| 2 | 水相1 | 有机相2 | 水相:有机相＝6:1 |
| 3 | 水相2 | 有机相1 | 水相:有机相＝6:1 |
| 4 | 水相2 | 有机相2 | 水相:有机相＝6:1 |
| 5 | 水相3 | 有机相1 | 水相:有机相＝6:1 |
| 6 | 水相3 | 有机相2 | 水相:有机相＝6:1 |
混合液立刻用pH7.4的PBS溶液稀释50-100倍，并使用切向流过滤法除去溶液中有机成分并浓缩至mRNA浓度约50μg/mL，得到包裹mRNA的脂质纳米颗粒，即为呼吸道合胞病毒核酸疫苗。
实施例3疫苗载体的优化
利用荧光素酶作为报告基因，通过活体荧光成像技术研究不同疫苗载体的配方在小鼠体内递送编码荧光素酶基因的mRNA的效率，并检测不同复合制剂(制备方法参见实施例2)的理化指标，结果如表3所示。
表3中，1～3号制剂水相采用的缓冲液成分与表2中水相1～3相同；有机相成分与表2中有机相1中有脂质成分相同，总脂质浓度为8mg/mL，水相:有机相＝4:1。
表3.不同水相制备制剂理化指标对比
结果表明，柠檬酸盐作为缓冲液时，包封率最高；乙酸钠为缓冲液时次之；磷酸盐作为缓冲液时，包封率最低。对柠檬酸盐和乙酸钠作为水相缓冲液进一步研究。表4中，1～10号制剂水相成分与表2中水相1、水相2相同；有机相成分与表2中有机相1、有机相2相同；调节水相与有机相比例，使各候选疫苗Lipid:mRNA质量比为20:1或40:1。
表4.不同复合制剂的理化指标及其在小鼠体内表达报告基因的效果
结果表明，提高配方胆固醇的含量有利于提高mRNA在体内的表达效率，脂质与mRNA质量比为40:1时有利于增加呼吸道合胞病毒mRNA在脂质纳米颗粒中的包封率，从而使其具有更高的稳定性；柠檬酸盐缓冲液作为水相溶剂时，表面包封率更高。因此综合考虑mRNA包封率以及mRNA体内递送效率等因素，7号、10号配方均可作为候选疫苗，用于后续呼吸道合胞病毒mRNA疫苗的研究。
实施例4中和抗体滴度及细胞免疫测定
mRNA选用实施例1中的含有RSV F-1和RSV F-5mRNA进行小鼠免疫实验，疫苗载体分别选取：脂质纳米颗粒应用实施例3中7号配方制备，每只小鼠按照10μg的给药量注射，注射体积为100μL/只。阴性对照为等体积生理盐水注射液。以上各种疫苗制剂在第0天、28天分两次给药，最后一次给药后14天检测小鼠血清呼吸道合胞病毒中和抗体滴度及细胞免疫，结果如下表5、表6所示：
表5RSV A型免疫应答反应
| 检测项目 | 阴性对照 | 7号配方制备疫苗 |
| 中和抗体(50％neutralizing titer NT<sub>50</sub>) | 4 | 23893±20672 |
| IL-2(％of CD4 T cells) | 0％ | 0.6±0.17 |
| TNF-α(％of CD4 T cells) | 0％ | 1.62±0.51 |
| IFN-γ(％of CD4 T cells) | 0％ | 2.65±0.81 |
| 阳性率 | 0％ | 100％ |
表6RSV B型免疫应答反应
| 检测项目 | 阴性对照 | 7号配方制备疫苗 |
| 中和抗体(50％neutralizing titer NT<sub>50</sub>) | 4 | 32768±24576 |
| IL-2(％of CD4 T cells) | 0％ | 1.03±0.43 |
| TNF-α(％of CD4 T cells) | 0％ | 2.16±0.74 |
| IFN-γ(％of CD4 T cells) | 0％ | 3.03±0.98 |
| 阳性率 | 0％ | 100％ |
从表中结果可以看出，两次注射后脂质纳米颗粒制剂可诱导产生抗呼吸道合胞病毒中和抗体及细胞免疫反应。
导致VERD效应产生的主要原因是疫苗不能有效诱导产生中和抗体，而非中和性抗体能够促进病毒的感染，加重疾病。本发明疫苗可以有效产生中和性抗体；此外，本发明提供的疫苗可以诱导机体产生细胞免疫应答的细胞因子TNF-α以及IFN-γ，而研究中发现，传统的灭活及亚单位疫苗不能产生细胞免疫相关细胞因子，因此本发明提供的mRNA疫苗能够避免VERD效应。
实施例5免疫持久性
应用实施例3中7号配方制备得到的呼吸道合胞病毒疫苗。将制备好的疫苗制剂用于棉鼠的免疫实验，给药量为10μg/只，给药体积为100μL/只。阴性对照为等体积生理盐水注射液。在第0天、28天分两次次通过肌肉注射途径接种，第二次给药后检测棉鼠体内中和抗体滴度变化结果如图2所示。从图中可以看出，给药后6个月内，本发明提供的核酸疫苗发现能够有效诱导产生中和抗体滴度。实施例4、5数据说明本发明技术优于现有技术。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 浙江君怡生物科技有限公司
<120> 呼吸道合胞病毒mRNA疫苗及其制备方法和应用
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Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro
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Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro
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Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile
50 55 60
Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu
85 90 95
Met Gln Asn Thr Pro Ala Val Asn Asn Arg Ala Arg Arg Glu Ala Pro
100 105 110
Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser
115 120 125
Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val
130 135 140
Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu
145 150 155 160
Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Gln Leu Thr Asn Lys
165 170 175
Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val
180 185 190
Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn
195 200 205
Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln
210 215 220
Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn
225 230 235 240
Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu
245 250 255
Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys
260 265 270
Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile
275 280 285
Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro
290 295 300
Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro
305 310 315 320
Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg
325 330 335
Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe
340 345 350
Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp
355 360 365
Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr
370 375 380
Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr
385 390 395 400
Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys
405 410 415
Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile
420 425 430
Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp
435 440 445
Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly
450 455 460
Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro
465 470 475 480
Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn
485 490 495
Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu
500 505 510
Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn Ile Met Ile Thr
515 520 525
Ala Ile Ile Ile Val Ile Ile Val Val Leu Leu Ser Leu Ile Ala Ile
530 535 540
Gly Leu Leu Leu Tyr Cys Lys Ala Lys Asn Thr Pro Val Thr Leu Ser
545 550 555 560
Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Lys
565 570
Claims (11)
1.编码呼吸道合胞病毒F蛋白的核酸，包括I)～IV)中至少一种：
I)、具有如SEQ ID NO:1或SEQ ID NO:2所示核苷酸序列的核酸；
II)、在I)所述的片段中取代、缺失或添加一个或多个核苷酸的核酸；
III)、与如I所示的核苷酸序列具有至少70％同源性的序列且编码呼吸道合胞病毒F蛋白的核酸；
IV)、与I)～III)中任一项部分互补或完全互补的核酸。
2.核糖核酸片段，其包括权利要求1所述的核酸，还包括5’-帽结构、5’-UTR、3’-UTR和3’-端PolyA中至少一种。
3.根据权利要求2所述的核糖核酸片段，其特征在于，所述：
5’-帽结构选自dmCAP、mCAP、tmCAP或ARCA中至少一种；
5’-UTR选自X-GLOBIN或A-GLOBIN；
3’-UTR选自X-GLOBIN、A-GLOBIN或脂氧合酶基因；
3’端PolyA的长度为20～100。
4.根据权利要求2或3所述的核糖核酸片段，其特征在于，由5’端至3’端依次包括：
X-GLOBIN 5’-UTR、SEQ ID NO:1所示序列的核酸、X-GLOBIN 3’-UTR和长度为50的PolyA；
或包括A-GLOBIN 5’-UTR、SEQ ID NO:1所示序列的核酸、A-GLOBIN 3’-UTR和长度为40的PolyA；
或包括X-GLOBIN 5’-UTR、SEQ ID NO:1所示序列的核酸、脂氧合酶基因3’-UTR和长度为30的PolyA；
或包括A-GLOBIN 5’-UTR、SEQ ID NO:1所示序列的核酸、脂氧合酶基因3’-UTR和长度为70的PolyA；
或包括X-GLOBIN 5’-UTR、SEQ ID NO:2所示序列的核酸、X-GLOBIN 3’-UTR和长度为80的PolyA；
或包括A-GLOBIN 5’-UTR、SEQ ID NO:2所示序列的核酸、A-GLOBIN 3’-UTR和长度为50的PolyA。
5.根据权利要求4所述的核糖核酸片段，其特征在于，
X-GLOBIN 5’-UTR的核酸序列如SEQ ID NO:3所示；
A-GLOBIN 5’-UTR的核酸序列如SEQ ID NO:4所示；
X-GLOBIN 3’-UTR的核酸序列如SEQ ID NO:5所示；
A-GLOBIN 3’-UTR的核酸序列如SEQ ID NO:6所示；
脂氧合酶基因3’-UTR的核酸序列如SEQ ID NO:7所示。
6.权利要求1所述的核酸或权利要求2～5任一项所述的核糖核酸片段在制备防治RSV病毒感染的疫苗中的应用。
7.疫苗，其特征在于，包括权利要求2～5任一项所述的核糖核酸片段、疫苗载体和辅料。
8.根据权利要求7所述的疫苗，其特征在于，
所述疫苗载体由可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂制得；所述可质子化阳离子脂质选自DOTAP、DODMA、Dlin-MC3-DMA和DlinDMA中的至少一种；所述结构脂质选自胆固醇或胆固醇衍生物中的至少一种；所述辅助脂质选自DSPC、DOPC、DOPE和DOPS中的至少一种；所述表面活性剂选自PEG-DMG和PEG-DSPE中的至少一种；
所述辅料为盐或盐的水溶液；所述盐选自柠檬酸盐、乙酸盐或磷酸盐中至少一种。
9.根据权利要求7或8所述的疫苗，其特征在于，其pH值为7.0～8.0。
10.权利要求7～9任一项所述疫苗的制备方法，其包括：
将权利要求2～5任一项所述的核糖核酸片段溶于辅料水溶液，得到水相组分；
将可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂溶于有机溶液得到有机相组分；所述有机溶剂为无水乙醇、异丙醇或丙酮；
将水相组分和有机相组分混合得到疫苗；或者以辅料水溶液稀释有机相组分后经浓缩得到疫苗。
11.根据权利要求9所述的疫苗的制备方法，其特征在于，水相组分pH值为3.0～7.0，其中权利要求2～5任一项所述的核糖核酸片段在水相中的含量为0.01mg/mL～2.0mg/mL；所述疫苗载体与权利要求2～5任一项所述的核糖核酸片段的质量比为(1～60):1。
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