WO2023202711A1

WO2023202711A1 - Mrna vaccine based on novel coronavirus

Info

Publication number: WO2023202711A1
Application number: PCT/CN2023/089871
Authority: WO
Inventors: 岑山; 王静; 衣岽戎; 董翊洁
Original assignee: 仁景(苏州)生物科技有限公司; 仁景国际香港有限公司
Priority date: 2022-04-22
Filing date: 2023-04-21
Publication date: 2023-10-26
Also published as: CN116925195A

Abstract

The present disclosure relates to an mRNA vaccine based on a novel coronavirus, in particular to a mRNA vaccine for preventing or treating coronavirus infection, a synthesis method for the mRNA vaccine, and an RNA composition. In particular, the present disclosure relates to an mRNA vaccine for preventing coronavirus infection by inducing an effective coronavirus antigen-specific immune response. The present disclosure further describes a method for preparing the vaccine and immunological evaluation of the vaccine.

Description

一种基于新型冠状病毒的mRNA疫苗A novel coronavirus-based mRNA vaccine

技术领域Technical field

本公开涉及用于预防或治疗冠状病毒感染的领域，具体涉及新型冠状病毒SARS-CoV-2mRNA疫苗及其制备方法和应用。特别地，本公开涉及用于针对冠状病毒感染进行疫苗接种并诱导有效的冠状病毒抗原特异性免疫应答如抗体和/或T细胞应答的方法和药剂。这些方法和药剂特别可用于预防或治疗冠状病毒感染。本公开还描述了用于制备所述疫苗的方法及所述疫苗的免疫学评价情况。The present disclosure relates to the field of preventing or treating coronavirus infection, and specifically relates to the novel coronavirus SARS-CoV-2 mRNA vaccine and its preparation method and application. In particular, the present disclosure relates to methods and agents for vaccination against coronavirus infection and inducing effective coronavirus antigen-specific immune responses, such as antibody and/or T cell responses. These methods and agents are particularly useful in preventing or treating coronavirus infections. This disclosure also describes methods for preparing the vaccines and immunological evaluation of the vaccines.

背景技术Background technique

2019新型冠状病毒(SARS-CoV-2)是以前从未在人体中发现的冠状病毒新毒株，该病毒为第七个可以感染人的冠状病毒(CoV)。人感染SARS-CoV-2的潜伏期一般为1～14天，感染后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重病例中，感染可导致肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。世卫组织网站最新数据显示，截至2021年11月26日全球累计报告确诊病例259502031例，死亡病例5183003例。The 2019 novel coronavirus (SARS-CoV-2) is a new strain of coronavirus that has never been found in humans before. This virus is the seventh coronavirus (CoV) that can infect humans. The incubation period for human infection with SARS-CoV-2 is generally 1 to 14 days. Common signs after infection include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. The latest data from the WHO website shows that as of November 26, 2021, a total of 259,502,031 confirmed cases and 5,183,003 deaths have been reported globally.

新冠病毒是正义单链RNA((+)ssRNA)包膜病毒，编码4种结构蛋白：刺突蛋白(S)、包膜蛋白(E)、膜蛋白(M)和核壳蛋白(N)。S蛋白分为S1和S2两个子结构域，S1结构域负责识别病毒特异性受体并结合至宿主细胞，S2具有跨膜结构域，负责膜融合。SARS-CoV-2 Delta变异病毒株最早于2020年10月在印度发现，后已传播至少185个国家和地区，成为全球主要流行的病毒变异株，而基于早期流行株研发的新冠疫苗针对SARS-CoV-2 Delta变异病毒株的中和能力均存在显著下降。The new coronavirus is a positive-sense single-stranded RNA ((+)ssRNA) enveloped virus encoding four structural proteins: spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N). The S protein is divided into two sub-domains, S1 and S2. The S1 domain is responsible for recognizing virus-specific receptors and binding to host cells. S2 has a transmembrane domain and is responsible for membrane fusion. The SARS-CoV-2 Delta mutant virus strain was first discovered in India in October 2020. It has since spread to at least 185 countries and regions, becoming the main popular virus mutant strain in the world. The new coronavirus vaccine developed based on early epidemic strains targets SARS- The neutralizing ability of CoV-2 Delta variant strains was significantly reduced.

目前上市和在研的新型冠状病毒疫苗主要包括mRNA疫苗、灭活疫苗、腺病毒载体疫苗、DNA疫苗、重组蛋白疫苗等。mRNA疫苗是将体外转录的mRNA递送至细胞，翻译产生蛋白，进而激发机体的特异免疫应答。mRNA疫苗不同于其他的核酸疫苗，不需要入核仅在细胞质中完成表达，因此没有造成宿主基因组***突变的风险。此外，mRNA的快速、简易制备方法和低成本也是作为疫苗的优势之一，大大缩短了应对突发感染性疾病的反应时间，降低了防控成本。本发明的目的就是制备一种新型冠状病毒mRNA疫苗。New coronavirus vaccines currently on the market and under development mainly include mRNA vaccines, inactivated vaccines, adenovirus vector vaccines, DNA vaccines, recombinant protein vaccines, etc. mRNA epidemic Vaccines deliver in vitro-transcribed mRNA to cells, where they are translated to produce proteins, thereby stimulating the body's specific immune response. Unlike other nucleic acid vaccines, mRNA vaccines do not need to enter the nucleus and are only expressed in the cytoplasm, so there is no risk of insertional mutations in the host genome. In addition, the rapid, simple preparation method and low cost of mRNA are also one of the advantages of vaccines, which greatly shortens the response time to respond to sudden infectious diseases and reduces the cost of prevention and control. The purpose of the present invention is to prepare a new coronavirus mRNA vaccine.

发明内容Contents of the invention

鉴于S蛋白在宿主细胞识别和进入以及宿主免疫***诱导病毒中和抗体中的重要性，我们将SARS-CoV-2的S蛋白用于疫苗开发。更具体地，本发明提供的mRNA疫苗包括至少一种具有编码SARS-CoV-2 Delta突变株抗原的开放阅读框的RNA多核苷酸。SARS-CoV-2抗原包括刺突蛋白(S蛋白)及其变体。S蛋白的融合前构象对建立有效的免疫***至关重要，因此为开发更有针对性的mRNA疫苗，我们选取了Delta病毒株的S蛋白基因编码序列以及在此基础上进行的特定脯氨酸位点的替代变体，分别得到Delta S，Delta S-2P(K984P，V985P)和Delta S-6P(F815P，A890P，A897P，A940P，K984P，V985P)的抗原序列。Given the importance of the S protein in host cell recognition and entry and induction of virus-neutralizing antibodies by the host immune system, we used the S protein of SARS-CoV-2 for vaccine development. More specifically, the mRNA vaccine provided by the invention includes at least one RNA polynucleotide having an open reading frame encoding a SARS-CoV-2 Delta mutant strain antigen. SARS-CoV-2 antigens include spike protein (S protein) and its variants. The pre-fusion conformation of the S protein is crucial to establishing an effective immune system. Therefore, in order to develop a more targeted mRNA vaccine, we selected the S protein gene coding sequence of the Delta virus strain and the specific proline on this basis. The substitution variants of the sites resulted in the antigen sequences of Delta S, Delta S-2P (K984P, V985P) and Delta S-6P (F815P, A890P, A897P, A940P, K984P, V985P) respectively.

在一个方面，本发明提供了一种RNA，其包括编码SARS-CoV-2的抗原性多肽或者其免疫原性片段或变体的开放阅读框，其中所述抗原性多肽选自SARS-CoV-2的受体结合结构域、S蛋白、其变体或者它们的免疫原性片段，优选地所述SARS-CoV-2是SARS-CoV-2Delta变异病毒株。In one aspect, the invention provides an RNA comprising an open reading frame encoding an antigenic polypeptide of SARS-CoV-2 or an immunogenic fragment or variant thereof, wherein the antigenic polypeptide is selected from the group consisting of SARS-CoV-2 2's receptor binding domain, S protein, variants thereof or immunogenic fragments thereof, preferably the SARS-CoV-2 is a SARS-CoV-2 Delta variant strain.

在一些实施方案中，对于所述的RNA，其中所述抗原性多肽或者其免疫原性片段或变体包含SARS-CoV-2多肽或其变体的一个或多个免疫原性表位；例如，至少2个，至少3个，至少4个，至少5个，至少6个，至少7个，至少8个，至少9个，至少10个或更多个免疫原性表位；优选地，所述抗原性多肽或者其免疫原性片段或变体选自SARS-CoV-2 Delta变异病毒株的(全长)S蛋白，优选地是SARS-CoV-2 Delta变异病毒株的(全长)S蛋白变体，更优选地所述S蛋白变体选自Delta S-2P，其包含突变K984P和V985P，以及Delta S-6P，其包含突变F815P、A890P、A897P、A940P、K984P和V985P，In some embodiments, for the RNA, wherein the antigenic polypeptide or immunogenic fragment or variant thereof comprises one or more immunogenic epitopes of a SARS-CoV-2 polypeptide or variant thereof; e.g. , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more immunogenic epitopes; preferably, the The antigenic polypeptide or the immunogenic fragment or variant thereof is selected from the (full-length) S protein of the SARS-CoV-2 Delta variant virus strain, preferably Is a (full-length) S protein variant of the SARS-CoV-2 Delta variant strain, more preferably the S protein variant is selected from Delta S-2P, which contains mutations K984P and V985P, and Delta S-6P, which Contains mutations F815P, A890P, A897P, A940P, K984P and V985P,

优选地，所述抗原性多肽或者其免疫原性片段或变体包含SEQ ID NO：1、2或3的氨基酸17-1271的氨基酸序列，与SEQ ID NO：1、2或3的氨基酸17-1271的氨基酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的氨基酸序列；和/或Preferably, the antigenic polypeptide or immunogenic fragment or variant thereof comprises the amino acid sequence of amino acids 17-1271 of SEQ ID NO: 1, 2 or 3, and the amino acid sequence 17-1271 of SEQ ID NO: 1, 2 or 3. The amino acid sequence of 1271 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical; and/or

编码所述抗原性多肽或者其免疫原性片段或变体的所述RNA包含SEQ ID NO：4、5或6的核苷酸49-3813的核苷酸序列，与SEQ ID NO：4、5或6的核苷酸49-3813的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列。The RNA encoding the antigenic polypeptide or an immunogenic fragment or variant thereof includes the nucleotide sequence of nucleotides 49-3813 of SEQ ID NO: 4, 5 or 6, which is the same as SEQ ID NO: 4, 5 or the nucleotide sequence of nucleotides 49-3813 of 6 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

在一些实施方案中，所述编码SARS-CoV-2的抗原性多肽或者其免疫原性片段或变体的开放阅读框还包含分泌信号肽，所述分泌信号肽优选地通过N-末端融合至所述的抗原性多肽或者其免疫原性片段或变体，所述分泌信号肽优选地是S蛋白的分泌信号肽。优选地，所述分泌信号肽包含SEQ ID NO：1、2或3的氨基酸1-16的氨基酸序列，与SEQ ID NO：1、2或3的氨基酸1-16的氨基酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的氨基酸序列，或者SEQ ID NO：1、2或3的氨基酸1-16的氨基酸序列的功能片段或与SEQ ID NO：1、2或3的氨基酸1-16的氨基酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的氨基酸序列的功能片段；和/或In some embodiments, the open reading frame encoding an antigenic polypeptide of SARS-CoV-2 or an immunogenic fragment or variant thereof further comprises a secretion signal peptide, preferably N-terminally fused to The antigenic polypeptide or the immunogenic fragment or variant thereof, the secretion signal peptide is preferably the secretion signal peptide of S protein. Preferably, the secretion signal peptide comprises the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3, which is at least 99% identical to the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3. An amino acid sequence that is 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or a functional fragment of the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3 or is identical to SEQ ID NO: 1, 2 or 3. ID NO: A functional fragment with an amino acid sequence of at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of amino acids 1-16 of 1, 2 or 3; and / or

编码所述分泌信号肽的RNA包含SEQ ID NO：4、5或6的核苷酸1-48的核苷酸序列，与SEQ ID NO：4、5或6的核苷酸1-48的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列，或者SEQ ID NO：4、5或6的核苷酸1-48的核苷酸序列的片段或与SEQ ID NO：4、5或6的核苷酸1-48的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列的片段。The RNA encoding the secretion signal peptide comprises the nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4, 5 or 6, and the nucleic acid sequence of nucleotides 1-48 of SEQ ID NO: 4, 5 or 6. A nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity, or the nucleotides of SEQ ID NO: 4, 5 or 6 A fragment of the nucleotide sequence 1-48 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or a fragment of a nucleotide sequence that is 80% identical.

在该方面的一些优选实施方案中，所述RNA为mRNA、环状RNA(cRNA)和自复制RNA(saRNA)，优选地所述RNA适合多肽的细胞内表达。In some preferred embodiments of this aspect, the RNA is mRNA, circular RNA (cRNA) and self-replicating RNA (saRNA), preferably the RNA is suitable for intracellular expression of the polypeptide.

在一些实施方案中，所述RNA是修饰的RNA，其通过用修饰的尿苷残基取代一些或全部尿苷残基进行修饰，优选地所述修饰的尿苷是N1-甲基-假尿苷。In some embodiments, the RNA is a modified RNA modified by replacing some or all of the uridine residues with a modified uridine residue, preferably the modified uridine is N1-methyl-pseudouridine glycosides.

在优选的实施方案中，所述RNA还包括一个或多个针对RNA在稳定性和翻译效率方面的最大效力进行优化的结构元件，优选地所述结构元件包括：5’帽、5’UTR、3’UTR和polyA尾序列。In a preferred embodiment, the RNA also includes one or more structural elements optimized for maximum effectiveness of the RNA in terms of stability and translation efficiency. Preferably, the structural elements include: 5' cap, 5' UTR, 3'UTR and polyA tail sequence.

优选地，所述5’帽是或包含cap1结构；更优选地，所述5’帽是m7G(5’)ppp(5’)(2’-OMeA)pG。Preferably, the 5' cap is or contains a cap1 structure; more preferably, the 5' cap is m7G(5')ppp(5')(2'-OMeA)pG.

优选地，所述5′-UTR是人β-珠蛋白mRNA的5′-UTR序列，任选地具有优化的Kozak序列；更优选地，所述5’UTR包含SEQ ID NO：7的核苷酸序列，或与SEQ ID NO：7的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列，Preferably, the 5'-UTR is the 5'-UTR sequence of human β-globin mRNA, optionally with an optimized Kozak sequence; more preferably, the 5'UTR comprises the nucleoside of SEQ ID NO: 7 acid sequence, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 7,

优选地，其中所述3′-UTR是人β-珠蛋白mRNA的两个重复的3′-UTR；更优选地，所述3’UTR包含SEQ ID NO：8的核苷酸序列，或与SEQ ID NO：8的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列。Preferably, wherein the 3'-UTR is a two-repeat 3'-UTR of human β-globin mRNA; more preferably, the 3'UTR comprises the nucleotide sequence of SEQ ID NO: 8, or is identical to The nucleotide sequence of SEQ ID NO: 8 is a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

优选地，所述polyA尾序列包含至少50、至少60或至少100个A核苷酸；更优选地，所述polyA尾序列包含SEQ ID NO：9的核苷酸序列，或由SEQ ID NO：9的核苷酸序列组成。Preferably, the polyA tail sequence comprises at least 50, at least 60 or at least 100 A nucleotides; more preferably, the polyA tail sequence comprises the nucleotide sequence of SEQ ID NO: 9, or consists of SEQ ID NO: It consists of 9 nucleotide sequences.

在另一方面，本发明提供了一种组合物，其包含本文所述的RNA。In another aspect, the invention provides a composition comprising an RNA as described herein.

在一些实施方案中，所述组合物配制为或待配制为液体、固体或其组合，优选地所述组合物配制为或待配制为用于注射或其他给药方式，优选地所述组合物配制为或待配制为用于肌肉内注射。In some embodiments, the composition is formulated or to be formulated as a liquid, solid, or a combination thereof, preferably the composition is formulated or to be formulated for injection or other mode of administration, preferably the composition Formulated or to be formulated for intramuscular injection.

在本发明的一些实施方案的组合物中，所述RNA与蛋白和/或脂质复合，以产生用于施用的RNA-颗粒。In the compositions of some embodiments of the invention, the RNA is combined with proteins and/or lipids complex to produce RNA-particles for administration.

在本发明的进一步实施方案的组合物中，将所述RNA配制于脂质纳米颗粒中，所述脂质纳米颗粒包含阳离子/可电离的脂质、磷脂、胆固醇和聚乙二醇(PEG)-脂质；优选地所述脂质纳米颗粒包含((4-羟基丁基)氮烷二基)双(己烷-6，1-二基)双(2-癸酸己酯)(((4-hydroxybutyl)azanediyl)bis(hexane-6，1-diyl)bis(2-hexyldecanoate))、2-[(聚乙二醇)-2000]-N，N-双十四烷基乙酰胺(2-[(polyethyleneglyco1)-2000]-N，N-ditetradecylacetamide)、1，2-二硬脂酰基-sn-甘油-3-磷酸胆碱(1，2-Distearoyl-sn-glycero-3-phosphocholine)和胆固醇；更优选地所述脂质纳米颗粒包含SM-102、二硬脂酰基磷脂酰胆碱、胆固醇和DMG-PEG2000。In a composition of a further embodiment of the invention, the RNA is formulated in lipid nanoparticles comprising cationic/ionizable lipids, phospholipids, cholesterol and polyethylene glycol (PEG) - Lipid; preferably the lipid nanoparticles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)((( 4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 2-[(polyethylene glycol)-2000]-N, N-ditetradecyl acetamide (2 -[(polyethyleneglyco1)-2000]-N,N-ditetradecylacetamide), 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-Distaroyl-sn-glycero-3-phosphocholine) and Cholesterol; more preferably the lipid nanoparticles comprise SM-102, distearoylphosphatidylcholine, cholesterol and DMG-PEG2000.

在脂质纳米颗粒中，阳离子/可电离的脂质、磷脂、胆固醇和聚乙二醇(PEG)-脂质的摩尔比为(40-55)∶(10-15)∶(35-45)∶(0.5-2.5)，In lipid nanoparticles, the molar ratio of cationic/ionizable lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids is (40-55):(10-15):(35-45) ∶(0.5-2.5)，

优选阳离子/可电离的脂质、磷脂、胆固醇和聚乙二醇(PEG)-脂质的摩尔比为50∶10∶38.5∶1.5。A preferred molar ratio of cationic/ionizable lipid, phospholipid, cholesterol and polyethylene glycol (PEG)-lipid is 50:10:38.5:1.5.

在本发明的一些实施方案的组合物中，将所述RNA配制为或待配制为胶体；优选地，所述RNA配制为颗粒，在形成的胶体分散相中存在50％或更多、75％或更多或者85％或更多的RNA；更优选地所述颗粒通过使溶于水相中的RNA暴露于溶于有机相中的脂质形成，其中优选地所述有机相包含乙醇；还优选地，所述颗粒通过使溶于水相中的RNA暴露于分散于水相中的脂质形成，其中优选地所述分散于水相中的脂质形成脂质体。In the compositions of some embodiments of the invention, the RNA is formulated or to be formulated as a colloid; preferably, the RNA is formulated as particles and is present in 50% or more, 75% of the colloidal dispersed phase formed or more or 85% or more RNA; more preferably said particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase, wherein preferably said organic phase comprises ethanol; further Preferably, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dispersed in the aqueous phase, wherein preferably the lipids dispersed in the aqueous phase form liposomes.

在本发明的一些实施方案中，所述RNA以1μg-100μg每剂量的范围的量存在于所述组合物中。In some embodiments of the invention, the RNA is present in the composition in an amount ranging from 1 μg to 100 μg per dose.

在还另一方面，本发明提供了本文所述RNA或组合物在制备药物中的用途，所述药物为疫苗，所述药物进一步包含一种或多种药学上可接受的载剂、稀释剂和/或赋形剂。In yet another aspect, the present invention provides the use of the RNA or composition described herein in the preparation of a medicament, the medicament being a vaccine, the medicament further comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients.

在一些实施方案中，本发明的药物用于在受试者中诱导针对冠状病毒的免疫应答，优选地针对冠状病毒抗原的特异性免疫应答。In some embodiments, the medicaments of the present invention are used to induce response to coronavirus in a subject. An immune response to a coronavirus, preferably a specific immune response to a coronavirus antigen.

在一些实施方案中，本发明的药物用于冠状病毒感染的治疗或预防性治疗。In some embodiments, the medicaments of the invention are used for the treatment or prophylactic treatment of coronavirus infections.

在优选的实施方案中，所述冠状病毒为β冠状病毒，优选地所述冠状病毒为沙贝病毒(sarbecovirus)，更优选地所述冠状病毒为SARS-CoV-2，进一步优选地所述冠状病毒包括：新冠病毒原始毒株(GD108)、SARS-CoV-2 Alpha变异病毒株、SARS-CoV-2 Beta变异病毒株、SARS-CoV-2 Delta变异病毒株和SARS-CoV-2 Omicron变异病毒株。In a preferred embodiment, the coronavirus is a betacoronavirus, preferably the coronavirus is a sarbecovirus, more preferably the coronavirus is SARS-CoV-2, and further preferably the coronavirus Viruses include: original strain of new coronavirus (GD108), SARS-CoV-2 Alpha variant strain, SARS-CoV-2 Beta variant strain, SARS-CoV-2 Delta variant strain and SARS-CoV-2 Omicron variant virus strain.

在本发明的所述方面中，当所述RNA、组合物或药物给予人源细胞时，实现可检测的所述抗原性多肽或者其免疫原性片段或变体表达，并且优选地这种表达持续至少24小时或更长的时间段。In said aspects of the invention, when said RNA, composition or medicament is administered to cells of human origin, detectable expression of said antigenic polypeptide or immunogenic fragment or variant thereof is achieved, and preferably such expression Lasts for at least 24 hours or longer.

在本发明的所述方面中，施用所述RNA、组合物或药物在受试者中产生免疫效果，所述免疫效果包括产生SARS-CoV-2中和抗体和/或T细胞应答，特别是稳健的TH1型T细胞应答，优选地CD4+和/或CD8+T细胞应答。In such aspects of the invention, administration of said RNA, composition or medicament produces an immune effect in a subject, said immune effect comprising the production of SARS-CoV-2 neutralizing antibodies and/or T cell responses, in particular Robust TH1 type T cell response, preferably CD4+ and/or CD8+ T cell response.

在本发明的所述方面中，施用所述RNA、组合物或药物在受试者中产生免疫应答，优选地，所述免疫应答包括产生针对SARS-CoV-2刺突蛋白的S1亚基的结合抗体滴度，更优选地所述免疫应答包括产生针对SARS-CoV-2病毒的中和抗体滴度。In such aspects of the invention, administration of said RNA, composition or medicament produces an immune response in a subject, preferably said immune response comprises the production of an immune response directed against the S1 subunit of the SARS-CoV-2 spike protein. In conjunction with antibody titers, more preferably the immune response includes the production of neutralizing antibody titers against the SARS-CoV-2 virus.

在本发明的所述方面中，在将所述RNA、组合物或药物施用给受试者之后7天，受试者(例如小鼠)的血清显示针对所述开放阅读框编码的多肽的抗体的产生。In such aspects of the invention, 7 days after administration of said RNA, composition or medicament to the subject, the serum of the subject (e.g., a mouse) exhibits antibodies directed against a polypeptide encoded by said open reading frame of production.

在本发明的所述方面中，在将所述RNA、组合物或药物施用给受试者之后14天，受试者(例如小鼠)的血清显示病毒中和活性。In this aspect of the invention, the serum of the subject (eg, mouse) exhibits virus neutralizing activity 14 days after administration of the RNA, composition or medicament to the subject.

在本发明的所述方面中，所述受试者为哺乳动物，优选地所述受试者为小鼠，还优选地所述受试者为人类。In this aspect of the invention, the subject is a mammal, preferably the subject is a mouse, further preferably the subject is a human.

本发明还提供了一种制备疫苗的方法，包括将本文所述的RNA配制于脂质纳米颗粒中，所述脂质纳米颗粒包含阳离子可电离的脂质、磷脂、胆固醇和聚乙二醇(PEG)-脂质；优选地所述脂质纳米颗粒包含((4-羟基丁基)氮烷二基)双(己烷-6，1-二基)双(2-癸酸己酯)、2-[(聚乙二醇)-2000]-N，N-双十四烷基乙酰胺、1，2-二硬脂酰基-sn-甘油-3-磷酸胆碱和胆固醇；更优选地所述脂质纳米颗粒包含SM-102、二硬脂酰基磷脂酰胆碱(DSPC)、胆固醇和DMG-PEG2000。The invention also provides a method for preparing a vaccine, comprising formulating the RNA described herein in lipid nanoparticles, the lipid nanoparticles comprising cationic ionizable lipids. lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids; preferably the lipid nanoparticles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl ) bis(2-hexyl decanoate), 2-[(polyethylene glycol)-2000]-N,N-distetradecyl acetamide, 1,2-distearoyl-sn-glycerol-3 -Phosphocholine and cholesterol; more preferably the lipid nanoparticles comprise SM-102, distearoylphosphatidylcholine (DSPC), cholesterol and DMG-PEG2000.

本发明还提供了在受试者中诱导针对冠状病毒的免疫应答，优选地针对冠状病毒抗原的特异性免疫应答的方法。在另一方面，还提供了用于冠状病毒感染的治疗或预防性治疗的方法。优选地，所述方法包括向受试者施用本文所述的RNA、组合物或药物。优选地，所述受试者为哺乳动物，优选地所述受试者为小鼠，还优选地所述受试者为人类。The present invention also provides methods of inducing an immune response against a coronavirus, preferably a specific immune response against a coronavirus antigen, in a subject. In another aspect, methods for the treatment or prophylactic treatment of coronavirus infections are also provided. Preferably, the method includes administering to a subject an RNA, composition or medicament described herein. Preferably, the subject is a mammal, preferably the subject is a mouse, and further preferably the subject is a human.

在本发明所述方法的一些实施方案中，所述冠状病毒为β冠状病毒，优选地所述冠状病毒为沙贝病毒，更优选地所述冠状病毒为SARS-CoV-2，进一步优选地所述冠状病毒包括：新冠病毒原始毒株(GD108)、SARS-CoV-2 Alpha变异病毒株、SARS-CoV-2 Beta变异病毒株、SARS-CoV-2 Delta变异病毒株和SARS-CoV-2 Omicron变异病毒株。In some embodiments of the method of the present invention, the coronavirus is a beta coronavirus, preferably the coronavirus is Sabei virus, more preferably the coronavirus is SARS-CoV-2, and further preferably the coronavirus is SARS-CoV-2. The above-mentioned coronaviruses include: the original strain of the new coronavirus (GD108), the SARS-CoV-2 Alpha variant strain, the SARS-CoV-2 Beta variant strain, the SARS-CoV-2 Delta variant strain and the SARS-CoV-2 Omicron Mutated virus strains.

附图说明Description of the drawings

图1.LNP-mRNA在293T细胞中的表达。Figure 1. Expression of LNP-mRNA in 293T cells.

图2：新型冠状病毒mRNA疫苗在小鼠模型上的免疫学评价。Figure 2: Immunological evaluation of novel coronavirus mRNA vaccine in mouse model.

图3：新型冠状病毒mRNA疫苗在恒河猴模型上的免疫学评价。Figure 3: Immunological evaluation of the new coronavirus mRNA vaccine in the rhesus monkey model.

图4：新型冠状病毒mRNA疫苗对恒河猴免疫后的生理学评价。Figure 4: Physiological evaluation of rhesus monkeys after immunization with the new coronavirus mRNA vaccine.

图5：恒河猴肺部病理改变评分。Figure 5: Score of pathological changes in the lungs of rhesus monkeys.

具体实施方式Detailed ways

除非另外指出，否则本发明的实践将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和合成生物学等等的常规技术，其在本领域技术范围内。这样的技术在文献中有充分的解释： “Molecular Cloning：A Laboratory Manual，”第二版(Sambrook等人，1989)；“Oligonucleotide Synthesis”(M.J.Gait编，1984)；“Animal Cell Culture”(R.I.Freshney编，1987)；“Methods in Enzymology”(Academic Press，Inc.)；“Current Protocols in Molecular Biology”(F.M.Ausubel等人编，1987，以及定期更新)；“PCR：The Polymerase Chain Reaction，”(Mullis等人编，1994)；Singleton等人，Dictionary of Microbiology and Molecular Biology第二版，J.Wiley&Sons(New York，N.Y.1994)和March’s Advanced Organic Chemistry Reactions，Mechanisms and Structure第四版，John Wiley&Sons(New York，N.Y.1992)，为本领域技术人员提供了本申请中使用的许多术语的通用指南。Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, synthetic biology, and the like, which are within the skill of the art. Such techniques are well explained in the literature: "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (edited by MJGait, 1984); "Animal Cell Culture" (edited by RI Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (FMAusubel et al., 1987, and regularly updated); "PCR: The Polymerase Chain Reaction," (Mullis et al., 1994); Singleton et al., Dictionary of Microbiology and Molecular Biology, second edition, J. Wiley & Sons (New York, NY 1994) and March's Advanced Organic Chemistry Reactions, Mechanisms and Structure, fourth edition, John Wiley & Sons (New York, NY 1992), are provided in this application for those skilled in the art. A general guide to the many terms used.

除非另有定义，否则本文使用的所有技术和科学术语具有与本发明所属领域普通技术人员通常理解的相同的含义。为了本发明的目的，下文定义了下述术语。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the purposes of the present invention, the following terms are defined below.

冠词“一个/一种”和“该/所述”在本文中用于指一个/一种或超过一个/一种(即至少一个/一种)所述冠词的语法对象。替代(例如“或”)的使用应理解为意指替代方案中任一、两者或其任何组合。术语“和/或”应理解为意指替代方案中任一或两者。The articles "a" and "the" are used herein to refer to one or more than one (i.e. at least one) of the grammatical objects of the article. The use of an alternative (eg, "or") should be understood to mean either, both, or any combination of the alternatives. The term "and/or" should be understood to mean either or both of the alternatives.

如本文使用的，术语“约”或“大约”是指与参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度相比较，改变多达15％、10％、9％、8％、7％、6％、5％、4％、3％、2％或1％的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度。As used herein, the term "about" or "approximately" means a change of up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of quantity, level, value, quantity, frequency, percentage, scale, size, quantity, weight or length.

在本说明书全文，除非上下文另有要求，否则术语“包含”，“包括”、“含有”和“具有”应理解为暗示包括所述步骤或要素或者步骤组或要素组，但不排除任何其他步骤或要素或者步骤组或要素组。在特定实施方式中，术语“包含”，“包括”、“含有”和“具有”同义使用。Throughout this specification, unless the context otherwise requires, the terms "comprising", "including", "containing" and "having" shall be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other Step or element or group of steps or elements. In certain embodiments, the terms "comprising," "including," "containing," and "having" are used synonymously.

“由......组成”意指包括但限于在短语“由......组成”后的任何。因此，短语“由......组成”是指示所列出的要素是需要的或强制性的，并且没有其他要素是可以存在的。"Consisting of" is meant to include, but be limited to, anything following the phrase "consisting of." Therefore, the phrase "consisting of" indicates that the listed elements are required or mandatory, and And no other elements can exist.

”基本上由......组成”意指包括在短语“基本上由......组成”后列出的任何要素，并且限于不干扰或贡献于所列出的要素的公开内容中指定的活动或动作的其他要素。因此，短语“基本上由......组成”是指示所列出的要素是需要的或强制性的，但没有其他要素是任选的，并且取决于它们是否影响所列出的要素的活动或动作而可以存在或不存在。"Consisting essentially of" is meant to include any of the elements listed after the phrase "consisting essentially of" and is limited to not interfering with or contributing to the disclosure of the listed elements Other elements of the activity or action specified in the content. Thus, the phrase "consisting essentially of" is an indication that the listed elements are required or mandatory, but that no other elements are optional and depend on whether they affect the listed elements An activity or action that may or may not exist.

在本说明书全文，提到“一个实施方案”、“一些实施方案”、“一个具体的实施方案”等类似表述意指与所述实施方式结合描述的特定特征、结构或特性被包括在本发明的至少一个实施方式中。因此，前述短语在本说明书全文的各个地方的出现不一定全部指相同实施方式。此外，特定特征、结构或特性可以以任何合适方式在一个或多个实施方式中组合。Throughout this specification, reference to "one embodiment," "some embodiments," "a specific embodiment," and the like means that a particular feature, structure, or characteristic described in connection with the embodiment is included in the invention. in at least one embodiment. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.

如本文所用的，“突变体”和“变体”是指保留与原始序列的生物学活性相同或基本上相同的生物学活性的分子。该突变体或变体可以来自相同或不同的物种，或者可以是基于天然的分子或现有的分子的合成序列。在一些实施方式中，术语“突变体”和“变体”是指多肽具有的氨基酸序列与对应的野生型多肽至少相差一个氨基酸。例如，突变体和变体可以包含保守氨基酸取代：即用具有相似性质的氨基酸取代原有的对应氨基酸。保守取代可以是极性对极性氨基酸(甘氨酸(G，Gly)、丝氨酸(S，Ser)、苏氨酸(T，Thr)、酪氨酸(Y，Tyr)、半胱氨酸(C，Cys)、天冬酰胺(N，Asn)和谷氨酰胺(Q，Gln))；非极性对非极性氨基酸(丙氨酸(A，Ala)、缬氨酸(V，Val)、色氨酸(W，Trp)、亮氨酸(L，Leu)、脯氨酸(P，Pro)、甲硫氨酸(M，Met)、苯丙氨酸(F，Phe))；酸性对酸性氨基酸(天冬氨酸(D，Asp)、谷氨酸(E，Glu))；碱性对碱性氨基酸(精氨酸(R，Arg)、组氨酸(H，His)、赖氨酸(K，Lys))；带电荷氨基酸对带电荷氨基酸(天冬氨酸(D，Asp)、谷氨酸(E，Glu)、组氨酸(H，His)、赖氨酸(K，Lys)和精氨酸 (R，Arg))；和疏水对疏水性氨基酸(丙氨酸(A，Ala)、亮氨酸(ULeu)、异亮氨酸(I，Ile)、缬氨酸(V，Val)、脯氨酸(P，Pro)、苯丙氨酸(F，Phe)、色氨酸(W，Trp)和甲硫氨酸(M，Met))。在一些其他实施方式中，突变体或变体也可以包含非保守性取代。As used herein, "mutant" and "variant" refer to molecules that retain the same or substantially the same biological activity as that of the original sequence. The mutant or variant may be from the same or different species, or may be based on a natural molecule or a synthetic sequence of an existing molecule. In some embodiments, the terms "mutant" and "variant" refer to a polypeptide having an amino acid sequence that differs from the corresponding wild-type polypeptide by at least one amino acid. For example, mutants and variants may contain conservative amino acid substitutions: substitution of amino acids with similar properties for corresponding amino acids. Conservative substitutions can be polar to polar amino acids (glycine (G, Gly), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine (C, Cys), asparagine (N, Asn) and glutamine (Q, Gln)); non-polar versus non-polar amino acids (alanine (A, Ala), valine (V, Val), chrom Acid (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (M, Met), phenylalanine (F, Phe)); acidic versus acidic Amino acids (aspartic acid (D, Asp), glutamic acid (E, Glu)); basic versus basic amino acids (arginine (R, Arg), histidine (H, His), lysine (K, Lys)); charged amino acids versus charged amino acids (aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys) ) and arginine (R, Arg)); and hydrophobic pairs of hydrophobic amino acids (alanine (A, Ala), leucine (ULeu), isoleucine (I, Ile), valine (V, Val), proline amino acid (P, Pro), phenylalanine (F, Phe), tryptophan (W, Trp) and methionine (M, Met)). In some other embodiments, mutants or variants may also contain non-conservative substitutions.

在一些实施方式中，突变体或变体多肽可以具有约1、2、3、4、5、6、7、8、9、10、15、20、30、40、50、60、70、80、90、100个或更多个或前述数值中的任意两者构成的范围的氨基酸的置换、添加、***或缺失。与未改变的多肽相比，突变体或变体可以具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％或前述数值中的任意两者构成的范围的活性。In some embodiments, a mutant or variant polypeptide can have about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 , 90, 100 or more, or substitution, addition, insertion or deletion of amino acids in a range consisting of any two of the aforementioned values. A mutant or variant may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or any two of the aforementioned values.

多核苷酸或多肽与另一个多核苷酸或多肽具有一定的“序列同一性”或“相同性”百分比，意味着当比对两条序列时，该百分比的碱基或氨基酸相同并且在相同的相对位置。确定两个氨基酸序列或两个核苷酸序列的百分比相同性可以包括比对和比较两个序列中相应位置处的氨基酸残基或核苷酸。如果两个序列中的所有位置被相同的氨基酸残基或核苷酸占据，那么所述序列被认为是100％相同的。序列相同性可以以多种不同方式确定，例如，可以使用各种方法和计算机程序(例如，BLAST、T-COFFEE、MUSCLE、MAFFT等)对序列进行比对。A polynucleotide or polypeptide has a certain percentage of "sequence identity" or "identity" to another polynucleotide or polypeptide, meaning that when the two sequences are compared, that percentage of bases or amino acids are the same and in the same relative position. Determining the percent identity of two amino acid sequences or two nucleotide sequences may include aligning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. Two sequences are considered 100% identical if all positions in the sequences are occupied by the same amino acid residues or nucleotides. Sequence identity can be determined in a number of different ways, for example, sequences can be aligned using a variety of methods and computer programs (eg, BLAST, T-COFFEE, MUSCLE, MAFFT, etc.).

本发明一般包括受试者的免疫治疗，包括施用RNA，即疫苗RNA，其编码氨基酸即疫苗抗原，包含SARS-CoV-2 S蛋白或其免疫原性片段或变体。因此，疫苗抗原包含SARS-CoV-2 S蛋白的表位，用于在受试者中诱导针对冠状病毒S蛋白，特别是SARS-CoV-2 S蛋白的免疫应答。施用编码疫苗抗原的RNA以(在通过适当的靶细胞表达多核苷酸之后)提供抗原，用于诱导，即刺激、引发和/或扩大免疫应答，例如，抗体和/或免疫效应细胞，其靶向靶抗原(冠状病毒S蛋白，特别是SARS-CoV-2 S蛋白)或其加工产物。根据本公开诱导的免疫应答是B细胞介导的免疫应答，即抗体介导的免疫应答，特别是抗SARS-CoV-2免疫应答。The invention generally encompasses the immunotherapy of a subject comprising the administration of RNA, i.e., vaccine RNA, encoding amino acids, i.e., vaccine antigens, comprising the SARS-CoV-2 S protein or immunogenic fragments or variants thereof. Therefore, the vaccine antigen contains an epitope of the SARS-CoV-2 S protein and is used to induce an immune response in a subject against the coronavirus S protein, particularly the SARS-CoV-2 S protein. RNA encoding a vaccine antigen is administered to provide (after expression of the polynucleotide by an appropriate target cell) an antigen for inducing, i.e., stimulating, priming and/or amplifying an immune response, e.g., antibodies and/or immune effector cells, which target To the target antigen (coronavirus S protein, especially SARS-CoV-2 S protein) or its processed products. According to this The immune response induced by disclosure is a B cell-mediated immune response, that is, an antibody-mediated immune response, especially an anti-SARS-CoV-2 immune response.

本文描述的疫苗包含作为有效成分的单链RNA，其可以在进入接受者细胞后翻译为相应的蛋白。除了编码抗原序列的野生型、突变型或密码子优化的序列，RNA还可以包含一个或多个结构元件，这些结构元件针对RNA在稳定性和翻译效率方面的最大效力进行优化(5′帽、5′UTR、3′UTR、polyA尾)。CleanCap形成的m7G(5’)ppp(5’)(2’-OMeA)pG可以用作RNA药物物质5′-端的特定加帽结构。作为5′-UTR序列，可以使用人β-珠蛋白mRNA的5′-UTR序列，任选地具有优化的“Kozak序列”以提高翻译效率。3′-UTR可以是人β-珠蛋白mRNA的两个重复的3′-UTR。此外，可以使用长度为50-120个核苷酸的polyA尾序列。The vaccine described herein contains as an active ingredient single-stranded RNA, which can be translated into the corresponding protein after entering the recipient cells. In addition to wild-type, mutant, or codon-optimized sequences encoding the antigenic sequence, the RNA may also contain one or more structural elements that are optimized for maximum potency of the RNA in terms of stability and translation efficiency (5' cap, 5′UTR, 3′UTR, polyA tail). The m7G(5’)ppp(5’)(2’-OMeA)pG formed by CleanCap can be used as a specific capping structure at the 5’-end of RNA drug substances. As the 5'-UTR sequence, the 5'-UTR sequence of human β-globin mRNA can be used, optionally with an optimized "Kozak sequence" to improve translation efficiency. The 3'-UTR may be the two repeated 3'-UTR of human β-globin mRNA. Additionally, polyA tail sequences of 50-120 nucleotides in length can be used.

此外，可以将分泌信号肽(sec)优选以sec翻译为N末端标签的方式融合至抗原编码区。在一个实施方案中，sec对应于S蛋白的分泌信号肽。Furthermore, a secretion signal peptide (sec) can be fused to the antigen coding region, preferably in such a way that sec is translated into an N-terminal tag. In one embodiment, sec corresponds to the secretion signal peptide of the S protein.

本文描述的疫苗RNA可以与蛋白和/或脂质(优选脂质)复合，以产生用于施用的RNA-颗粒。如果使用不同RNA的组合，可以将RNA一起或分别与蛋白和/或脂质复合以产生用于施用的RNA-颗粒。The vaccine RNA described herein can be complexed with proteins and/or lipids (preferably lipids) to generate RNA-particles for administration. If a combination of different RNAs is used, the RNAs can be complexed together or separately with proteins and/or lipids to generate RNA-particles for administration.

在一方面，本发明涉及一种组合物或药物制品(medical preparation)，其包含编码氨基酸序列的RNA，所述氨基酸序列包含SARS-CoV-2 S蛋白或其免疫原性片段或变体。In one aspect, the invention relates to a composition or medical preparation comprising an RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof.

在一个实施方案中，包含SARS-CoV-2 S蛋白或其免疫原性片段或变体的氨基酸序列能够形成多聚体复合物，特别是三聚体复合物。为此，包含SARS-CoV-2 S蛋白或其免疫原性片段或变体的氨基酸序列可以包含允许形成多聚体复合物的结构域，特别是包含SARS-CoV-2 S蛋白或其免疫原性片段或变体的氨基酸序列的三聚体复合物。在一个实施方案中，允许形成多聚体复合物的结构域包含三聚结构域，例如，本文描述的三聚结构域。 In one embodiment, the amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof is capable of forming a multimeric complex, in particular a trimer complex. To this end, the amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof may comprise a domain that allows the formation of a multimeric complex, in particular comprising the SARS-CoV-2 S protein or an immunogen thereof. Trimeric complexes of amino acid sequences of sexual fragments or variants. In one embodiment, the domain that allows the formation of multimeric complexes comprises a trimerization domain, eg, a trimerization domain described herein.

在一个实施方案中，包含SARS-CoV-2 S蛋白或其免疫原性片段或变体的氨基酸序列由编码序列编码，与野生型编码序列相比，所述编码序列是密码子优化的和/或G/C含量增加，其中所述密码子优化和/或G/C含量增加优选地不改变编码的氨基酸序列的序列。In one embodiment, the amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof is encoded by a coding sequence that is codon-optimized and/or compared to a wild-type coding sequence. or an increase in G/C content, wherein said codon optimization and/or increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence.

在一个实施方案中，(i)编码SARS-CoV-2 S蛋白或其免疫原性片段或变体的RNA包含SEQ ID NO：4、5或6的核苷酸49-3813的核苷酸序列，与SEQ ID NO：4、5或6的核苷酸49-3813的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列；和/或In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof comprises the nucleotide sequence of nucleotides 49-3813 of SEQ ID NO: 4, 5 or 6 , having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 49-3813 of SEQ ID NO: 4, 5 or 6 the nucleotide sequence; and/or

(ii)SARS-CoV-2 S蛋白或其免疫原性片段或变体包含SEQ ID NO：1、2或3的氨基酸17-1271的氨基酸序列，与SEQ ID NO：1、2或3的氨基酸17-1271的氨基酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的氨基酸序列。(ii) SARS-CoV-2 S protein or an immunogenic fragment or variant thereof contains the amino acid sequence of amino acids 17-1271 of SEQ ID NO: 1, 2 or 3, and the amino acid sequence of SEQ ID NO: 1, 2 or 3 The amino acid sequence of 17-1271 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

在一个实施方案中，包含SARS-CoV-2 S蛋白、其免疫原性变体或者所述SARS-CoV-2 S蛋白或其免疫原性片段或变体的免疫原性片段的氨基酸序列包含分泌信号肽。In one embodiment, the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein, or an immunogenic fragment or variant thereof, comprises secreted signal peptide.

在一个实施方案中，所述分泌信号肽融合，优选通过N-末端融合，至SARS-CoV-2 S蛋白或其免疫原性片段或变体。In one embodiment, the secretion signal peptide is fused, preferably by N-terminal fusion, to the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof.

在一个实施方案中，(i)编码所述分泌信号肽的RNA包含SEQ ID NO：4、5或6的核苷酸1-48的核苷酸序列，与SEQ ID NO：4、5或6的核苷酸1-48的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列，或者SEQ ID NO：4、5或6的核苷酸1-48的核苷酸序列的片段或与SEQ ID NO：4、5或6的核苷酸1-48的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列的片段；和/或In one embodiment, (i) the RNA encoding the secretion signal peptide comprises a nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4, 5 or 6, identical to SEQ ID NO: 4, 5 or 6 A nucleotide sequence of nucleotides 1-48 that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to a nucleotide sequence, or SEQ ID NO: A fragment of the nucleotide sequence of nucleotides 1-48 of 4, 5 or 6 or has at least 99%, 98%, A fragment of a nucleotide sequence that is 97%, 96%, 95%, 90%, 85% or 80% identical; and/or

(ii)所述分泌信号肽包含SEQ ID NO：1、2或3的氨基酸1-16的氨基酸序列，与SEQ ID NO：1、2或3的氨基酸1-16的氨基酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的氨基酸序列，或者SEQ ID NO：1、2或3的氨基酸1-16的氨基酸序列的功能片段或与SEQ ID NO：1、2或3的氨基酸1-16的氨基酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的氨基酸序列的功能片段。(ii) The secretion signal peptide comprises the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3, and has at least 99%, An amino acid sequence that is 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or amino acids 1-16 of SEQ ID NO: 1, 2 or 3 A functional fragment of the acid sequence or is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3 A functional fragment of a sexual amino acid sequence.

在一个实施方案中，所述RNA是修饰的RNA，特别是稳定的mRNA。在一个实施方案中，所述RNA包含修饰的核苷，以代替尿苷。在一个实施方案中，所述修饰的核苷是N1-甲基-假尿苷(m1ψ)。In one embodiment, the RNA is modified RNA, in particular stabilized mRNA. In one embodiment, the RNA contains a modified nucleoside in place of uridine. In one embodiment, the modified nucleoside is N1-methyl-pseudouridine (mlψ).

在一个实施方案中，编码包含SARS-CoV-2 S蛋白或其免疫原性片段或变体的氨基酸序列的RNA包含5’UTR，其包含SEQ ID NO：7的核苷酸序列，或与SEQ ID NO：7的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列。In one embodiment, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO: 7, or the same sequence as SEQ ID NO: 7 The nucleotide sequence of ID NO: 7 is a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

在一个实施方案中，编码包含SARS-CoV-2 S蛋白或其免疫原性片段或变体的氨基酸序列的RNA包含3’UTR，其包含SEQ ID NO：8的核苷酸序列，或与SEQ ID NO：8的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列。In one embodiment, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof comprises a 3'UTR that comprises the nucleotide sequence of SEQ ID NO: 8, or is identical to SEQ ID NO: 8 The nucleotide sequence of ID NO: 8 is a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

在一个实施方案中，编码包含SARS-CoV-2 S蛋白或其免疫原性片段或变体的氨基酸序列的RNA包含polyA尾序列。在一个实施方案中，所述polyA尾序列包含至少100个核苷酸。在一个实施方案中，所述polyA尾序列包含SEQ ID NO：9的核苷酸序列，或由SEQ ID NO：9的核苷酸序列组成。In one embodiment, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof comprises a polyA tail sequence. In one embodiment, the polyA tail sequence contains at least 100 nucleotides. In one embodiment, the polyA tail sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 9.

在一个实施方案中，所述RNA或组合物配制为或待配制为液体、固体或其组合。在一个实施方案中，所述RNA或组合物配制为或待配制为用于注射或其他给药方式。在一个实施方案中，所述RNA或组合物配制为或待配制为用于肌肉内注射。In one embodiment, the RNA or composition is formulated or to be formulated as a liquid, solid, or a combination thereof. In one embodiment, the RNA or composition is or is to be formulated for injection or other administration. In one embodiment, the RNA or composition is or is to be formulated for intramuscular injection.

在一个实施方案中，所述RNA配制为或待配制为颗粒。在一个实施方案中，所述颗粒为脂质纳米颗粒(LNP)。In one embodiment, the RNA is formulated or to be formulated as particles. In one embodiment, the particles are lipid nanoparticles (LNPs).

在一个实施方案中，所述LNP颗粒包含(4-羟基丁基)氮烷二基)双(己烷-6，1-二基)双(2-癸酸己酯)(((4-hydroxybutyl)azanediyl)bis(hexane-6，1-diyl)bis(2-hexyldecanoate))、2-[(聚乙二醇)-2000]-N，N-双十四烷基乙酰胺(2-[(polyethylene glyc01)-2000]-N，N-ditetradecylacetamide)、1，2-二硬脂酰基-sn-甘油-3-磷酸胆碱(1，2-Distearoyl-sn-glycero-3-phosphocholine)和胆固醇。In one embodiment, the LNP particles comprise (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)((4-hydroxybutyl )azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 2-[(polyethylene glycol)-2000]-N, N-ditetradecyl acetamide (2-[( polyethylene glyc01)-2000]-N,N-ditetradecylacetamide), 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-Distearoyl-sn-glycero-3-phosphocholine) and cholesterol.

在一个实施方案中，所述RNA配制为或待配制为胶体。在一个实施方案中，所述RNA配制为或待配制为颗粒，形成胶体的分散相。在一个实施方案中，所述分散相中存在50％或更多、75％或更多或者85％或更多的RNA。在一个实施方案中，所述RNA配制或待配制为包含RNA和脂质的颗粒。在一个实施方案中，所述颗粒通过使溶于水相中的RNA暴露于溶于有机相中的脂质形成。在一个实施方案中，所述有机相包含乙醇。在一个实施方案中，所述颗粒通过使溶于水相中的RNA暴露于分散于水相中的脂质形成。在一个实施方案中，所述分散于水相中的脂质形成脂质体。In one embodiment, the RNA is formulated or to be formulated as a colloid. In one embodiment, the RNA is formulated or to be formulated as particles forming a colloidal dispersed phase. In one embodiment, 50% or more, 75% or more, or 85% or more RNA is present in the dispersed phase. In one embodiment, the RNA is formulated or to be formulated as particles comprising RNA and lipids. In one embodiment, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase. In one embodiment, the organic phase contains ethanol. In one embodiment, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dispersed in the aqueous phase. In one embodiment, the lipids dispersed in the aqueous phase form liposomes.

在一个实施方案中，所述RNA为mRNA、环状RNA(cRNA)和自复制RNA(saRNA)。In one embodiment, the RNA is mRNA, circular RNA (cRNA), and self-replicating RNA (saRNA).

在一个实施方案中，所述组合物或药物制品为药物组合物。在一个实施方案中，所述组合物或药物制品为疫苗。在一个实施方案中，所述药物组合物进一步包含一种或多种药学上可接受的载剂、稀释剂和/或赋形剂。In one embodiment, the composition or pharmaceutical preparation is a pharmaceutical composition. In one embodiment, the composition or pharmaceutical preparation is a vaccine. In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.

在一方面，本发明涉及本文描述的组合物或药物制品，其用于制药用途。在一个实施方案中，所述制药用途包括在受试者中诱导针对冠状病毒的免疫应答。在一个实施方案中，所述制药用途包括冠状病毒感染的治疗或预防性治疗。在一个实施方案中，所述冠状病毒为β冠状病毒。在一个实施方案中，所述冠状病毒为沙贝病毒(sarbecovirus)。在一个实施方案中，所述冠状病毒为SARS-CoV-2。在进一步优选的实施方案中，所述冠状病毒包括：新冠病毒原始毒株(GD108)、SARS-CoV-2 Alpha变异病毒株、SARS-CoV-2 Beta变异病毒株、SARS-CoV-2 Delta变异病毒株和SARS-CoV-2 Omicron变异病毒株In one aspect, the invention relates to a composition or pharmaceutical preparation described herein for pharmaceutical use. In one embodiment, the pharmaceutical use includes inducing an immune response against coronavirus in a subject. In one embodiment, the pharmaceutical use includes treatment or prophylactic treatment of coronavirus infection. In one embodiment, the coronavirus is a betacoronavirus. In one embodiment, the coronavirus is a sarbecovirus. In one embodiment, the coronavirus is SARS-CoV-2. In further preferred embodiments, the coronavirus includes: the original strain of the new coronavirus (GD108), the SARS-CoV-2 Alpha variant strain, the SARS-CoV-2 Beta variant strain, and the SARS-CoV-2 Delta variant Virus strains and SARS-CoV-2 Omicron variant strains

在一方面，本发明涉及一种在受试者中诱导针对冠状病毒的免疫应答的方法，所述方法包括向受试者施用包含编码氨基酸序列的 RNA的组合物，所述氨基酸序列包含SARS-CoV-2 S蛋白或其免疫原性片段或变体。In one aspect, the invention relates to a method of inducing an immune response against a coronavirus in a subject, the method comprising administering to the subject a protein comprising an encoding amino acid sequence. A composition of RNA, the amino acid sequence comprising SARS-CoV-2 S protein or an immunogenic fragment or variant thereof.

在一个实施方案中，所述方法为针对冠状病毒进行疫苗接种的方法。在一个实施方案中，所述方法为用于冠状病毒感染的治疗或预防性治疗的方法。在一个实施方案中，所述受试者为小鼠。在一个实施方案中，所述冠状病毒为β冠状病毒。在一个实施方案中，所述冠状病毒为沙贝病毒(sarbecovirus)。在一个实施方案中，所述冠状病毒为SARS-CoV-2。在进一步优选的实施方案中，所述冠状病毒包括：新冠病毒原始毒株(GD108)、SARS-CoV-2 Alpha变异病毒株、SARS-CoV-2 Beta变异病毒株、SARS-CoV-2 Delta变异病毒株和SARS-CoV-2 Omicron变异病毒株In one embodiment, the method is a method of vaccination against a coronavirus. In one embodiment, the method is a method for the treatment or prophylactic treatment of coronavirus infection. In one embodiment, the subject is a mouse. In one embodiment, the coronavirus is a betacoronavirus. In one embodiment, the coronavirus is a sarbecovirus. In one embodiment, the coronavirus is SARS-CoV-2. In further preferred embodiments, the coronavirus includes: the original strain of the new coronavirus (GD108), the SARS-CoV-2 Alpha variant strain, the SARS-CoV-2 Beta variant strain, and the SARS-CoV-2 Delta variant Virus strains and SARS-CoV-2 Omicron variant strains

在一方面，本发明涉及本文描述的组合物或药物制品，其用于本文描述的方法。In one aspect, the invention relates to a composition or pharmaceutical preparation described herein for use in the methods described herein.

其中，本公开证实包含脂质纳米颗粒包裹的mRNA的组合物可以在根据方案(包括施用至少一个剂量的疫苗组合物)向小鼠施用之后7天内实现血清中针对表位的可检测的抗体滴度，所述mRNA编码SARS-CoV-2编码的多肽(例如，SARS-CoV-2编码的S蛋白)的至少一部分(例如，是或包含表位)。Among other things, the present disclosure demonstrates that a composition comprising lipid nanoparticle-encapsulated mRNA can achieve detectable antibody drops in serum against an epitope within 7 days after administration to mice according to a protocol that includes administration of at least one dose of the vaccine composition. Degree, the mRNA encodes at least a portion (e.g., is or includes an epitope) of a polypeptide encoded by SARS-CoV-2 (e.g., the S protein encoded by SARS-CoV-2).

本公开记录了其中mRNA内的核苷酸经修饰的提供的组合物(例如，包含脂质纳米颗粒包裹的mRNA的组合物，所述mRNA编码SARS-CoV-2编码的多肽(例如，SARS-CoV-2编码的S蛋白)的至少一部分(例如，是或包含表位))和/或涉及这类组合物的提供的方法的特征在于不存在内在佐剂作用，或者与具有未修饰的结果的其他可比组合物(或方法)相比减少的内在佐剂作用。可选地或额外地，在一些实施方案中，这类组合物(或方法)的特征在于它们诱导抗体应答和/或CD4+T细胞应答。在涉及修饰的核苷酸的一些实施方案中，这类修饰的核苷酸可以存在于例如3’UTR序列、抗原编码序列和/或5’UTR序列中。在一些实施方案中，修饰的核苷酸是或包括一个或多个修饰的尿嘧啶残基。 The present disclosure records provided compositions (e.g., compositions comprising lipid nanoparticle-encapsulated mRNA encoding a polypeptide encoded by SARS-CoV-2 (e.g., SARS- At least a portion of the S protein encoded by CoV-2 (e.g., is or contains an epitope)) and/or provided methods involving such compositions are characterized by the absence of intrinsic adjuvant effects, or are associated with unmodified results Reduced intrinsic adjuvant effect compared to other comparable compositions (or methods). Alternatively or additionally, in some embodiments, such compositions (or methods) are characterized in that they induce an antibody response and/or a CD4+ T cell response. In some embodiments involving modified nucleotides, such modified nucleotides may be present, for example, in the 3'UTR sequence, the antigen-encoding sequence, and/or the 5'UTR sequence. In some embodiments, the modified nucleotide is or includes one or more modified uracil residues.

其中，本公开记录了提供的组合物(例如，包含脂质纳米颗粒包裹的mRNA的组合物，所述mRNA编码SARS-CoV-2编码的多肽(例如，SARS-CoV-2编码的S蛋白)的至少一部分(例如，是或包含表位))和/或方法的特征在于编码的多肽(例如，SARS-CoV-2编码的蛋白(如S蛋白))，在一些实施方案中，所述部分可以是或包含其表位)的持续表达。例如，在一些实施方案中，这类组合物和/或方法的特征在于，当给予人源细胞时，它们实现可检测的多肽表达，并且在一些实施方案中，这种表达持续至少24小时或更长的时间段。Therein, the present disclosure records providing compositions (e.g., compositions comprising lipid nanoparticle-encapsulated mRNA encoding a SARS-CoV-2-encoded polypeptide (e.g., SARS-CoV-2-encoded S protein) At least a portion of (e.g., is or includes an epitope)) and/or the method is characterized by an encoded polypeptide (e.g., a protein encoded by SARS-CoV-2 (e.g., S protein)), and in some embodiments, said portion may be or comprise an epitope thereof). For example, in some embodiments, such compositions and/or methods are characterized in that they achieve detectable polypeptide expression when administered to cells of human origin, and in some embodiments, such expression persists for at least 24 hours or longer time period.

阅读本公开的本领域技术人员会进一步理解它描述了各种mRNA构建体，其包含全长SARS-CoV-2刺突蛋白的核酸序列(例如，包括其中这样的编码的SARS-CoV-2刺突蛋白可以包含至少一个或多个氨基酸取代的实施方案，例如，如本文描述的脯氨酸取代，和/或其中mRNA序列针对受试者(例如，哺乳动物，例如，人)进行密码子优化的实施方案)。更进一步，阅读本公开的这样的普通技术人员会理解它描述了包含编码全长SARS-CoV-2刺突蛋白的核酸序列的某些mRNA构建体的特定特征和/或优点。不希望受任何特定理论的束缚。在一些实施方案中，阅读本公开的普通技术人员会理解提供的编码全长SARS-CoV-2 S蛋白的mRNA构建体，可以对于用作或用于免疫原性组合物(例如，疫苗)实现如本文描述的免疫效果(例如，产生SARS-CoV-2中和抗体，和/或T细胞应答(例如，CD4+和/或CD8+T细胞应答))特别有用和/或有效。Those of skill in the art who read this disclosure will further understand that it describes various mRNA constructs that comprise the nucleic acid sequence of the full-length SARS-CoV-2 spike protein (e.g., including the SARS-CoV-2 spike protein encoded therein). The spike protein may comprise embodiments in which at least one or more amino acid substitutions are made, e.g., a proline substitution as described herein, and/or in which the mRNA sequence is codon-optimized for the subject (e.g., mammal, e.g., human) implementation). Further, one of ordinary skill reading this disclosure will understand that it describes certain features and/or advantages of certain mRNA constructs comprising nucleic acid sequences encoding the full-length SARS-CoV-2 spike protein. There is no wish to be bound by any particular theory. In some embodiments, one of ordinary skill reading this disclosure will understand that the provided mRNA constructs encoding the full-length SARS-CoV-2 S protein can be implemented for use in or for use in immunogenic compositions (e.g., vaccines) Immune effects (eg, generation of SARS-CoV-2 neutralizing antibodies, and/or T cell responses (eg, CD4+ and/or CD8+ T cell responses)) as described herein are particularly useful and/or effective.

在一些实施方案中，本公开提供包含编码全长SARS-CoV-2 S蛋白(例如，具有一个或多个氨基酸取代的全长SARS-CoV-2 S蛋白)的开放阅读框的RNA(例如，mRNA)，所述RNA适合多肽的细胞内表达。在一些实施方案中，这样的RNA可以配制于脂质纳米颗粒(例如，本文描述的脂质纳米颗粒)中。In some embodiments, the disclosure provides an RNA (e.g., mRNA), which RNA is suitable for intracellular expression of polypeptides. In some embodiments, such RNA can be formulated in lipid nanoparticles (eg, lipid nanoparticles described herein).

在一些实施方案中，本文提供的免疫原性组合物可以包含SARS-CoV-2多肽或其变体的多个(例如，至少2个或更多个，包括，例如，至少3个，至少4个，至少5个，至少6个，至少7个，至少8个，至少9个，至少10个等)免疫原性表位。在一些这样的实施方案中，这样的多个免疫原性表位可以由单一RNA(例如，mRNA)编码。不希望受任何特定理论的束缚，在一些实施方案中，当考虑SARS-CoV-2变体的遗传多样性时，提供的多表位免疫原性组合物(包括，例如，编码全长SARS-CoV-2刺突蛋白的那些)可以特别用于提供针对多种病毒变体的保护和/或可以提供更大的机会来开发多样化和/或稳健的中和抗体和/或T细胞应答，特别是特别稳健的TH1型T细胞(例如，CD4+和/或CD8+T细胞)应答。In some embodiments, immunogenic compositions provided herein can comprise multiple (e.g., at least 2 or more, including, For example, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.) immunogenic epitopes. In some such embodiments, such multiple immunogenic epitopes may be encoded by a single RNA (eg, mRNA). Without wishing to be bound by any particular theory, in some embodiments, when considering the genetic diversity of SARS-CoV-2 variants, multi-epitope immunogenic compositions provided (including, for example, encoding full-length SARS- those of the CoV-2 spike protein) may be particularly useful in providing protection against multiple viral variants and/or may provide greater opportunities to develop diverse and/or robust neutralizing antibody and/or T cell responses, In particular, a particularly robust TH1 type T cell (eg, CD4+ and/or CD8+ T cell) response.

在一些实施方案中，本公开记录了提供的组合物和/或方法的特征在于它们用单一施用实现一种或多种特定治疗结果(例如，如本文描述的有效免疫应答和/或编码的SARS-CoV-2 S蛋白的可检测表达)。In some embodiments, the present disclosure documents that compositions and/or methods are provided that are characterized in that they achieve one or more specific therapeutic outcomes (e.g., an effective immune response as described herein and/or encoded SARS) with a single administration. - detectable expression of CoV-2 S protein).

在一些实施方案中，免疫应答可以包括产生针对SARS-CoV-2刺突蛋白的S1亚基的结合抗体滴度。在一些实施方案中，免疫应答可以包括产生针对SARS-CoV-2病毒的中和抗体滴度。In some embodiments, the immune response can include generating binding antibody titers directed against the S1 subunit of the SARS-CoV-2 spike protein. In some embodiments, the immune response can include the production of neutralizing antibody titers against the SARS-CoV-2 virus.

在一些实施方案中，中和抗体滴度是(例如，已建立为)相对于适当对照(例如，未疫苗接种的对照小鼠)所观察到的足以减少或阻断病毒与疫苗接种的小鼠血清结合的滴度。In some embodiments, the neutralizing antibody titer is (e.g., established to be) sufficient to reduce or block virus compared to vaccinated mice compared to an appropriate control (e.g., unvaccinated control mice). Serum binding titers.

实施例Example

在下文中，将通过实施例详细描述本发明。然而，在此提供的实施例仅用于说明目的，并不用于限制本发明。Hereinafter, the present invention will be described in detail through examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the invention.

下述实施例所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are all conventional methods unless otherwise specified.

下述实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

实施例1.新型冠状病毒mRNA疫苗的制备Example 1. Preparation of novel coronavirus mRNA vaccine

1.S蛋白基因合成和载体构建 1.S protein gene synthesis and vector construction

1.1 S蛋白基因合成及优化1.1 S protein gene synthesis and optimization

此新型冠状病毒mRNA疫苗的目标抗原为Delta变异病毒株的S蛋白全长，其基因的编码序列见GenBank：QWU18818.1，并在此基础上进行不同位点的组合突变，分别获得Delta S-2P(K984P、V985P)和Delta S-6P(F815P、A890P、A897P、A940P、K984P、V985P)。根据氨基酸序列，反推编码的核苷酸，由基因合成公司经过人源密码子优化及相关酶切位点排除后进行合成。The target antigen of this new coronavirus mRNA vaccine is the full length of the S protein of the Delta mutant virus strain. The coding sequence of its gene can be found in GenBank: QWU18818.1. On this basis, combinatorial mutations at different sites are carried out to obtain Delta S- 2P (K984P, V985P) and Delta S-6P (F815P, A890P, A897P, A940P, K984P, V985P). Based on the amino acid sequence, the encoded nucleotides were deduced and synthesized by a gene synthesis company after optimization of human codons and elimination of relevant enzyme cutting sites.

1.2载体构建1.2 Vector construction

将合成的上述S序列进行BamHI和AscI双酶切，酶切产物进行1％琼脂糖凝胶电泳切胶回收后，进行连接反应，将依次带有5’UTR、目的基因、3’UTR及polyA尾序列的pUC57-luc载体中的luc编码序列替换为S基因序列，形成pUC57-S重组质粒。利用卡那抗生素进行单克隆平板初筛后，小提质粒并通过BamHI和AscI限制性内切酶酶切法和DNA测序进行阳性克隆验证。The synthesized S sequence was double digested with BamHI and AscI. The digested product was recovered by 1% agarose gel electrophoresis and then ligated. The 5'UTR, target gene, 3'UTR and polyA were sequentially digested. The luc coding sequence in the pUC57-luc vector of the tail sequence is replaced with the S gene sequence to form the pUC57-S recombinant plasmid. After preliminary screening of monoclonal plates using kana antibiotics, the plasmid was mini-prepared and positive clones were verified by BamHI and AscI restriction endonuclease digestion and DNA sequencing.

2.mRNA的制备2. Preparation of mRNA

2.1质粒线性化2.1 Plasmid linearization

质粒包含T7启动子、5’UTR、ORF、3’UTR以及polyA尾序列，在polyA尾序列最后一个A后面有一个SapI酶切位点。用限制性内切酶SapI线性化含有目的基因的质粒，反应体系如表1所示，37℃酶切3h。The plasmid contains the T7 promoter, 5'UTR, ORF, 3'UTR and polyA tail sequence. There is a SapI restriction site after the last A of the polyA tail sequence. Use the restriction endonuclease SapI to linearize the plasmid containing the target gene. The reaction system is shown in Table 1 and digest at 37°C for 3 hours.

表1质粒线性化酶切体系
Table 1 Plasmid linearization enzyme digestion system

取2μL酶切产物进行1％琼脂糖凝胶电泳，检测质粒是否被完全线性化。利用PCR产物回收试剂盒(康维世纪)纯化线性化质粒。Take 2 μL of the digested product and conduct 1% agarose gel electrophoresis to check whether the plasmid is completely linearized. The linearized plasmid was purified using PCR product recovery kit (Converse).

2.2体外转录2.2 In vitro transcription

以线性化重组质粒为模板进行体外转录，按如下体系(表2)加入各组分(以20μL反应体系为例)，混匀后37℃反应2h。 Use the linearized recombinant plasmid as a template for in vitro transcription. Add each component according to the following system (Table 2) (taking a 20 μL reaction system as an example), mix and react at 37°C for 2 hours.

表2体外转录体系
Table 2 In vitro transcription system

转录反应结束后，加入1μL DNase I，37℃反应15min，加入15μL反应终止液。After the transcription reaction is completed, add 1 μL DNase I, react at 37°C for 15 minutes, and add 15 μL reaction stop solution.

2.3 RNA纯化2.3 RNA purification

向体外转录反应体系中加入1/3体积的7.5M LiCl(使其终浓度为2.5M)，-20℃放置30min。12000g离心15min，RNA沉淀在底部，弃掉上清。加入1mL 70％乙醇清洗RNA，12000g离心5min，弃掉上清。晾干后加入50μL无RNA酶的水溶解沉淀，并使用紫外分光光度计进行mRNA定量，得到加帽的体外转录mRNA。Add 1/3 volume of 7.5M LiCl to the in vitro transcription reaction system (making the final concentration 2.5M), and place it at -20°C for 30 minutes. Centrifuge at 12000g for 15 minutes, RNA will precipitate at the bottom, and the supernatant will be discarded. Add 1mL of 70% ethanol to wash the RNA, centrifuge at 12000g for 5 minutes, and discard the supernatant. After drying, add 50 μL of RNase-free water to dissolve the precipitate, and use a UV spectrophotometer for mRNA quantification to obtain capped in vitro-transcribed mRNA.

3.LNP包载3.LNP package

将mRNA原液分散于20mM醋酸溶液(pH 6.0)，使终浓度为200μg/mL。通过控制水相端和油相端注射泵的流速，使mRNA与脂质混合物(配方见表3)按3∶1的体积比进行T混流混合，得到LNP包载的mRNA。然后通过超滤进行替液和浓缩。用Ribogreen法测定脂质体mRNA的浓度，游离RNA直接取样测定，总RNA用5％OTG裂解后测定，计算包封率，均在90％以上。用马尔文ZetaSizer粒径仪测量脂质体mRNA的粒径，均为60nm左右。Disperse the mRNA stock solution in 20mM acetic acid solution (pH 6.0) to make the final concentration 200μg/mL. By controlling the flow rates of the syringe pumps at the water phase end and the oil phase end, the mRNA and lipid mixture (see Table 3 for the recipe) are mixed in a T-flow at a volume ratio of 3:1 to obtain LNP-encapsulated mRNA. Liquid replacement and concentration are then performed by ultrafiltration. The concentration of liposome mRNA was measured using the Ribogreen method, free RNA was directly sampled and measured, and total RNA was measured after cleavage with 5% OTG. The encapsulation efficiency was calculated, and all were above 90%. The particle size of liposome mRNA was measured using a Malvern ZetaSizer particle size analyzer, and both were around 60 nm.

表3脂质混合物配方
Table 3 Lipid mixture formula

4.S目标抗原的表达鉴定4. Expression identification of S target antigen

将293T细胞接种到六孔板中，24h后在细胞培养基中加入2μg LNP包载的mRNA，进行细胞内表达。24h后收集细胞，每孔细胞沉淀加入100μL 1×蛋白上样缓冲液于100℃金属浴中裂解变性30min。蛋白样品储存于-20℃或者-80℃。通过免疫印迹检测S蛋白的表达：取8μL蛋白裂解液进行10％SDS-PAGE电泳，分离蛋白样品。使用PVDF膜75V恒压湿法转膜1.2h后，5％脱脂牛奶室温封闭1h。先后与一抗(抗SARS-CoV-2 S蛋白兔抗，1∶1000)和二抗(山羊抗兔，1∶5000)进行孵育，最后ECL显色。结果如图1所示，Delta S、Delta S-2P和Delta S-6P三个mRNA疫苗在细胞水平均能检测到S蛋白的表达。293T cells were seeded into a six-well plate, and 2 μg of LNP-encapsulated mRNA was added to the cell culture medium 24 hours later for intracellular expression. Collect the cells after 24 hours, add 100 μL of 1× protein loading buffer to each well of the cell pellet, and lyse and denature in a 100°C metal bath for 30 minutes. Protein samples were stored at -20°C or -80°C. Detect the expression of S protein by immunoblotting: take 8 μL of protein lysate and perform 10% SDS-PAGE electrophoresis to separate the protein samples. Use PVDF membrane to wet transfer at 75V constant pressure for 1.2 hours, then block with 5% skim milk at room temperature for 1 hour. It was incubated with primary antibody (anti-SARS-CoV-2 S protein rabbit antibody, 1:1000) and secondary antibody (goat anti-rabbit, 1:5000), and finally ECL color development. The results are shown in Figure 1. Delta S, Delta S-2P and Delta S-6P mRNA vaccines can all detect the expression of S protein at the cellular level.

实施例2.不同新型冠状病毒mRNA疫苗在小鼠模型上的免疫学评价Example 2. Immunological evaluation of different novel coronavirus mRNA vaccines on mouse models

1.小鼠的免疫接种和血清分离1. Immunization of Mice and Isolation of Serum

使用本发明的mRNA疫苗对SPF级雌性BALB/c小鼠(6-8周龄)进行注射免疫，免疫方案如图2A所示。免疫组别为LNP对照组、不同mRNA疫苗组。本次实验用血清为二次免疫后14天血清。SPF grade female BALB/c mice (6-8 weeks old) were injected and immunized using the mRNA vaccine of the present invention, and the immunization scheme is shown in Figure 2A. The immune groups are LNP control group and different mRNA vaccine groups. The serum used in this experiment was the serum 14 days after the second immunization.

2.疫苗免疫血清对SARS-CoV-2假病毒的中和活性检测2. Detection of neutralizing activity of vaccine immune serum against SARS-CoV-2 pseudovirus

在稳定过表达SARS-CoV-2主要受体ACE2(Angiotensin-converting enzyme 2)的Hela-ACE2细胞系(记载于“Liu X，Wei L，Xu F，Zhao F，Huang Y，Fan Z，Mei S，Hu Y，Zhai L，Guo J，Zheng A，Cen S，Liang C，Guo F.SARS-CoV-2spike protein-induced cell fusion activates the cGAS-STING pathway and the interferon response.Sci Signal.2022Apr 12；15(729)：eabg8744.doi：10.1126/scisignal.abg8744.Epub 2022 Apr 12.PMID：35412852.”)中检测血清针对SARS-CoV-2 Delta、Omicron毒株假型病毒的中和活性。假型病毒是S(Spike)蛋白包装的以慢病毒为核心，萤火虫荧光素酶报告基因的单轮感染假型病毒。将待检测的血清用DMEM培养基进行2倍系列稀释，共稀释8个梯度，从1∶128起稀释至1∶16384。在P2实验室中用DMEM培养基将假病毒稀释至1.5×10⁴TCID₅₀/mL。取各梯度的血清与稀释好的病毒液混合，37℃，5％CO₂温箱中孵育1小时。将孵育好的病毒血清混合物按每孔100μl加入到预先接种的Hela-ACE2细胞板中，置于37℃，5％CO₂温箱中培养。48小时后，测定萤火虫荧光素酶活性，计算假病毒感染性，以抑制50％荧光素酶活性的血清最高稀释度的倒数为终点效价。利用GraphPad Prism 8软件对Delta-S(6P)组与其它各组间的显著性差异进行双尾学生t检验统计学分析(*p＜0.05vs.Delta-S(6P))。In the Hela-ACE2 cell line that stably overexpresses the major SARS-CoV-2 receptor ACE2 (Angiotensin-converting enzyme 2) (documented in "Liu X, Wei L, Xu F, Zhao F, Huang Y, Fan Z, Mei S , Hu Y, Zhai L, Guo J, Zheng A, Cen S, Liang C, Guo F. SARS-CoV-2spike protein-induced cell fusion activates the cGAS-STING pathway and the interferon response. Sci Signal. 2022Apr 12; 15 (729): eabg8744.doi: 10.1126/scisignal.abg8744.Epub 2022 Apr 12. PMID: 35412852.") to detect the neutralizing activity of serum against SARS-CoV-2 Delta and Omicron strain pseudotypes. Pseudotyped virus is a single-round infection pseudotyped virus packaged by S (Spike) protein with lentivirus as the core and firefly luciferase reporter gene. The serum to be tested was serially diluted 2-fold with DMEM culture medium, and a total of 8 gradients were diluted, starting from 1:128 to 1:16384. Pseudovirus was diluted to 1.5 × 10 ⁴ TCID ₅₀ /mL in DMEM medium in the P2 laboratory. Take the serum of each gradient and mix it with the diluted virus liquid, and incubate it in a 37°C, 5% _CO2 incubator for 1 hour. Add 100 μl of the incubated virus serum mixture per well to the pre-seeded Hela-ACE2 cell plate, and place it in a 37°C, 5% _CO2 incubator. After 48 hours, the firefly luciferase activity was measured, and the infectivity of the pseudovirus was calculated. The reciprocal of the highest dilution of the serum that inhibited 50% of the luciferase activity was used as the end-point titer. GraphPad Prism 8 software was used to conduct a two-tailed Student's t test statistical analysis on the significant differences between the Delta-S (6P) group and other groups (*p<0.05 vs. Delta-S (6P)).

参见图2B中的结果表明，与Delta S和Delta S-2P相比，Delta S-6P免疫的小鼠血清对Delta株假病毒具有更高的中和活性。Delta S-6P免疫两次后的小鼠血清诱导的针对Omicron株假病毒的中和活性(PNT50)达到992，是Delta S和Delta S-2P疫苗的2-4倍。See results in Figure 2B showing that serum from mice immunized with Delta S-6P had higher neutralizing activity against Delta strain pseudovirus compared with Delta S and Delta S-2P. The neutralizing activity (PNT50) induced by mouse sera against Omicron strain pseudoviruses after two immunizations with Delta S-6P reached 992, which is 2-4 times that of Delta S and Delta S-2P vaccines.

3.疫苗免疫血清对SARS-CoV-2真病毒的中和抗体检测3. Detection of neutralizing antibodies against SARS-CoV-2 true virus in vaccine immune serum

在Vero-E6细胞上检测免疫小鼠血清针对SARS-CoV-2的5个毒株(流行株(即新冠原始毒株(GD108))、Alpha、Beta、Delta和Omicron株)的中和抗体效价。将待检测的血清56℃灭活30分钟。用DMEM培养基进行2倍系列稀释，从1∶8开始稀释。在P3实验室中根据病毒的原始滴度，用DMEM培养基将病毒稀释至工作浓度。取稀释好的病毒液与各稀释梯度的血清混合，37℃，5％CO₂温箱中孵育1小时。将孵育好的病毒血清混合物按每孔100μl加入到悬浮Vero-E6细胞板中，置于37℃，5％CO₂温箱中培养，72小时后通过判定细胞CPE来计算血清中和抗体效价(EC₅₀)。同时评估被免疫恒河猴的血清中和抗体交叉保护效果。该实验是在中国医学科学院医学生物学研究所的P3实验室进行的。利用GraphPad Prism 8软件对Delta S-6P组与其它各组间的显著性差异进行双尾学生t检验统计学分析(**p＜0.01vs.Delta S-6P；*p＜0.05vs.Delta S-6P)。The neutralizing antibody efficacy of immunized mouse sera against five strains of SARS-CoV-2 (epidemic strain (i.e., the original strain of COVID-19 (GD108)), Alpha, Beta, Delta and Omicron strains) was detected on Vero-E6 cells. price. The serum to be tested was inactivated at 56°C for 30 minutes. Perform 2-fold serial dilutions in DMEM culture medium, starting from 1:8. In the P3 laboratory, the virus was diluted to working concentration in DMEM medium according to the original titer of the virus. Mix the diluted virus liquid with the serum of each dilution gradient, and incubate for 1 hour at 37°C in a 5% _CO2 incubator. Add 100 μl of the incubated virus serum mixture into the suspended Vero-E6 cell plate per well, place it in a 37°C, 5% _CO2 incubator, and calculate the serum neutralizing antibody titer by determining the cell CPE after 72 hours. (EC ₅₀ ). The cross-protective effect of serum neutralizing antibodies in immunized rhesus monkeys was also evaluated. The experiment was conducted in the P3 laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences. GraphPad Prism 8 software was used to perform two-tailed Student's t test statistical analysis on the significant differences between the Delta S-6P group and other groups (**p<0.01vs.Delta S-6P; *p<0.05vs.Delta S -6P).

图2C中的结果显示，这三种疫苗产生的中和抗体对多种毒株具有交叉保护作用。其中Delta S-6P免疫的小鼠血清对这五种毒株的感染均表现出很强的中和活性，尤其是针对Omicron毒株的中和活性明显好于Delta S和Delta S-2P。The results in Figure 2C show that the neutralizing antibodies produced by these three vaccines have cross-protective effects against multiple strains. Among them, the serum of mice immunized with Delta S-6P showed strong neutralizing activity against the infection of these five strains, especially the neutralizing activity against the Omicron strain. Significantly better than Delta S and Delta S-2P.

实施例3.不同新型冠状病毒mRNA疫苗在恒河猴模型上的免疫学评价Example 3. Immunological evaluation of different novel coronavirus mRNA vaccines on rhesus monkey models

1.恒河猴的免疫接种1. Immunization of rhesus monkeys

利用Delta S-6P mRNA疫苗对恒河猴进行肌肉注射免疫，免疫时间点为D0(一免)、D21(二免)。免疫组别为LNP对照组、低剂量组(30μg)mRNA疫苗、高剂量组(100μg)mRNA疫苗。在第二次免疫后28天进行攻毒。3组实验动物均使用IND株(Delta)(毒种批21V05P0345/21V05P0346/21V05P0347，使用滴度1×10⁶TCID₅₀/mL)，通过鼻腔滴鼻+气管注射接种各500μL的方式进行攻毒，每只猴子攻毒量为1×10⁶TCID₅₀。免疫方案如图3A所示。Delta S-6P mRNA vaccine was used to immunize rhesus monkeys by intramuscular injection, and the immunization time points were D0 (first vaccination) and D21 (second vaccination). The immune groups are LNP control group, low-dose group (30 μg) mRNA vaccine, and high-dose group (100 μg) mRNA vaccine. Challenge was performed 28 days after the second immunization. The three groups of experimental animals all used the IND strain (Delta) (virus seed batch 21V05P0345/21V05P0346/21V05P0347, using a titer of 1×10 ⁶ TCID ₅₀ /mL), and were challenged by inoculating 500 μL of each via nasal instillation + tracheal injection. The amount of virus attacked by each monkey is 1×10 ⁶ TCID ₅₀ . The immunization protocol is shown in Figure 3A.

2.血清中抗原特异性IgG抗体检测2. Detection of antigen-specific IgG antibodies in serum

在免疫前(D0)及免疫后D7、D14、D21(二免)、D27、D35采血，分离对照组、低剂量组和高剂量组所有12只动物的血清，采用ELISA(enzyme-linked immunosorbent assay)法测定血清中抗原特异性结合抗体IgG水平。使用原始株的S蛋白保守性相对较高的NTD蛋白包被96孔酶标板，包被浓度为100ng/孔，体积100μL，2-8℃包被过夜。次日，用洗涤液(PBST)洗涤3次后，使用封闭液(含2％BSA的PBST溶液)25℃封闭2小时。进一步，将梯度稀释后的血清样品按100μL/孔加入96孔板，25℃孵育2小时。血清稀释倍数从1∶100开始，2倍倍比稀释；孵育结束后，用PBST洗涤3次，加入HRP耦连的抗猴IgG，25℃孵育1小时。最后，使用PBST洗涤5次，按100μL/孔加入TMB，避光显色，再加入100μL/孔终止液。酶标仪上读取数据，以610nm作为参比波长，450nm读取OD值，进而计算血清IgG滴度。Blood was collected before immunization (D0) and on D7, D14, D21 (secondary immunization), D27, and D35 after immunization, and the serum of all 12 animals in the control group, low-dose group, and high-dose group was separated, and ELISA (enzyme-linked immunosorbent assay) was used ) method to measure the level of antigen-specific binding antibody IgG in serum. Use the NTD protein with relatively high S protein conservation of the original strain to coat a 96-well microplate, with a coating concentration of 100ng/well, a volume of 100μL, and coating overnight at 2-8°C. The next day, after washing three times with washing solution (PBST), use blocking solution (PBST solution containing 2% BSA) for 2 hours at 25°C. Further, the serially diluted serum samples were added to a 96-well plate at 100 μL/well, and incubated at 25°C for 2 hours. The serum dilution ratio started from 1:100 and was diluted 2 times; after the incubation, it was washed three times with PBST, HRP-coupled anti-monkey IgG was added, and incubated at 25°C for 1 hour. Finally, wash 5 times with PBST, add 100 μL/well of TMB, protect from light for color development, and then add 100 μL/well of stop solution. Read the data on a microplate reader, use 610nm as the reference wavelength, read the OD value at 450nm, and then calculate the serum IgG titer.

图3B中的结果显示：低剂量组和高剂量组的所有动物经mRNA疫苗免疫后均在D14开始产生高水平结合抗体。对照组所有4只动物均无新冠病毒特异性抗体产生。 The results in Figure 3B show that all animals in the low-dose group and the high-dose group began to produce high-level binding antibodies on D14 after being immunized with the mRNA vaccine. All four animals in the control group did not produce new coronavirus-specific antibodies.

3.疫苗的中和效价检测3. Vaccine neutralization titer testing

采集实验猴血清，按照与实施例1相同的实验方法使用新冠病毒真病毒进行中和效价检测。The serum of experimental monkeys was collected, and the neutralization titer of the new coronavirus true virus was tested according to the same experimental method as in Example 1.

图3C-G中的结果显示：高剂量组和低剂量组二免后的实验猴血清均检测到一定程度的中和抗体水平，其中免疫组针对原始株和Delta毒株的中和效价较高。对照组均没有检测到中和抗体。The results in Figure 3C-G show that a certain degree of neutralizing antibody levels were detected in the serum of experimental monkeys after the second immunization in both the high-dose group and the low-dose group. Among them, the neutralizing titers of the immunized group against the original strain and the Delta strain were higher. high. No neutralizing antibodies were detected in the control group.

进一步的实验用血清为二次免疫后14天血清。按照与实施例1相同的实验方法检测血清针对SARS-CoV-2原始株、Delta、Omicron BA.1、Omicron BA.2.12.1、Omicron BA.4/5毒株假型病毒的中和活性。图3H所示的结果显示，疫苗免疫组针对原始株和Delta株假病毒有很强的中和效果。高低剂量组针对Omicron株的NT₅₀值如下：对于BA.1株分别为～826和～1172；对于BA.2.12.1株分别为～570和～905；对于BA4/5株分别为～364和～463，这些都表明该疫苗具有在恒河猴中诱导广谱中和抗体的潜力。The serum used in further experiments was the serum 14 days after the second immunization. The neutralizing activity of the serum against the original strain of SARS-CoV-2, Delta, Omicron BA.1, Omicron BA.2.12.1, and Omicron BA.4/5 pseudotyped viruses was tested according to the same experimental method as in Example 1. The results shown in Figure 3H show that the vaccine immune group has a strong neutralizing effect against the original strain and Delta strain pseudovirus. The NT ₅₀ values for the Omicron strains in the high and low dose groups are as follows: ∼826 and ∼1172 for the BA.1 strain, respectively; ∼570 and ∼905 for the BA.2.12.1 strain, respectively; and ∼364 and ∼364 for the BA4/5 strain, respectively. ~463, which indicate that the vaccine has the potential to induce broadly neutralizing antibodies in rhesus monkeys.

针对高剂量组和低剂量组二免后第14天的PBMCs样本进行分析，采用Mabtech公司的商品化检测试剂盒(货号为：PBMC IFN-γELISpot Assay：3421M-4APW-2；PBMC IL-2 ELISpot Assay：3421M-4AST-2)，利用ELISpot(enzyme-linked immune absorbent spot)法检测SARS-CoV-2的原始株、Delta株和Omicron株的Spike蛋白刺激后，抗原特异性T细胞分泌IFN-γ和IL-2的能力。图3I中的结果显示：疫苗组在二免后可显著激活细胞免疫应答。针对新冠病毒Delta株，Omicron株及原始株产生的细胞免疫应答水平无显著剂量组内差别。For analysis of PBMCs samples from the high-dose group and the low-dose group on the 14th day after the second vaccination, Mabtech's commercial detection kit (product number: PBMC IFN-γELISpot Assay: 3421M-4APW-2; PBMC IL-2 ELISpot Assay: 3421M-4AST-2), using ELISpot (enzyme-linked immune absorbent spot) method to detect the secretion of IFN-γ by antigen-specific T cells after stimulation by the Spike protein of the original strain, Delta strain and Omicron strain of SARS-CoV-2. and IL-2 abilities. The results in Figure 3I show that the vaccine group can significantly activate cellular immune responses after the second immunization. There was no significant intra-dose difference in the level of cellular immune response against the new coronavirus Delta strain, Omicron strain and original strain.

实施例4.新型冠状病毒mRNA疫苗对恒河猴免疫后的生理学评价Example 4. Physiological evaluation of rhesus monkeys after immunization with novel coronavirus mRNA vaccine

1.动物病毒载量检测1. Animal viral load detection

攻毒前及攻毒后第1，3，5，7天对恒河猴进行麻醉，采集鼻拭子、咽拭子、肛拭子。拭子样本处理：鼻、咽、肛拭子用800μl Trizol裂解，取其中200μL使用自动核酸提取仪提取RNA模板，使用 qRT-PCR(一步法)测定SARS-CoV-2基因组RNA水平。Rhesus monkeys were anesthetized before challenge and on days 1, 3, 5, and 7 after challenge, and nasal swabs, throat swabs, and anal swabs were collected. Swab sample processing: Nasal, pharyngeal, and anal swabs were lysed with 800 μl Trizol, and 200 μL was used to extract RNA template using an automatic nucleic acid extractor. qRT-PCR (one-step method) determines SARS-CoV-2 genomic RNA levels.

图4A中的结果显示，与对照组相比，Delta S-6P免疫的恒河猴鼻拭子、咽拭子、肛拭子的SARS-CoV-2基因组RNA水平显著降低。The results in Figure 4A show that compared with the control group, the levels of SARS-CoV-2 genomic RNA in nasal swabs, throat swabs, and anal swabs of rhesus monkeys immunized with Delta S-6P were significantly reduced.

2.动物肺部病毒载量检测2. Detection of viral load in animal lungs

攻毒后第7天解剖观察肺部大体病变情况，每只猴子分别取左肺(上、中、下叶)和右肺(上、中、下)组织。每叶肺采用多点(6个点)随机取样方式，取样点贯穿整片肺叶，混合称重，总重量接近100mg，以800μL Trizol进行组织匀浆，取其中400μL提取RNA模板，利用qRT-PCR(一步法)检测病毒载量。On the 7th day after the challenge, the gross lesions of the lungs were dissected and observed. The left lung (upper, middle, and lower lobes) and right lung (upper, middle, and lower) tissues were taken from each monkey. Each lung lobe adopts a multi-point (6 points) random sampling method. The sampling points run through the entire lung lobe. The sampling points are mixed and weighed. The total weight is close to 100mg. Tissue homogenization is performed with 800μL Trizol. 400μL is taken to extract the RNA template and qRT-PCR is used. (One-step method) Detection of viral load.

图4B中的结果显示，与对照组相比，在接种Delta S-6P疫苗的实验猴肺叶组织病毒载量明显降低，这表明SARS-CoV-2病毒复制受到强烈抑制。此外，100μg Delta S-6P免疫组整体显示出比30μg剂量更好的病毒清除效果。The results in Figure 4B show that compared with the control group, the viral load in the lung lobe tissue of experimental monkeys vaccinated with Delta S-6P vaccine was significantly reduced, indicating that SARS-CoV-2 virus replication was strongly inhibited. In addition, the 100 μg Delta S-6P immunized group showed overall better virus clearance than the 30 μg dose.

3.动物肺部病理学分析3. Pathological analysis of animal lungs

每只恒河猴每叶肺组织(左上、左中、左下、右上、右中、右下，共6叶肺)切片均经苏木素-伊红(H&E)染色和全图扫描。对恒河猴肺部炎性、肺部结构变化和出血等进行分级，各项指标的评分标准见下表4。The sections of each rhesus monkey's lung tissue (upper left, middle left, lower left, upper right, middle right, lower right, total 6 lobes) were stained with hematoxylin and eosin (H&E) and scanned in full. Pulmonary inflammation, lung structural changes, and bleeding in rhesus monkeys were graded. The scoring standards for each indicator are shown in Table 4 below.

表4.恒河猴肺部病理改变评分表
Table 4. Rhesus monkey lung pathological changes scoring table

根据评分表对每只恒河猴肺组织病理的每叶肺的病理图谱进行评分，评分主要针对新冠肺炎的主要病理特征：肺间隔增厚或实变程度、肺间隔出血程度、炎性细胞浸润程度、血管血栓、尘细胞分布面积等特征性指标进行评定，各项指标评分的合计数即为病理评分。选择至少5个视野进行评分，所有肺叶的病理评分的平均数即为这只猴子整个肺部的综合病理评分结果。图5的结果表明，低剂量组和高剂量组肺部病理损伤评分显著低于对照组(p<0.01)。The pathological atlas of each lobe of each rhesus monkey's lung tissue was evaluated according to the scoring table. Score, the score is mainly based on the main pathological characteristics of COVID-19: the degree of lung septal thickening or consolidation, the degree of lung septal bleeding, the degree of inflammatory cell infiltration, vascular thrombus, dust cell distribution area and other characteristic indicators are evaluated. Each indicator is scored. The total number is the pathological score. At least 5 fields of view were selected for scoring, and the average of the pathological scores of all lung lobes was the comprehensive pathological score of the monkey's entire lungs. The results in Figure 5 show that the lung pathological damage scores of the low-dose group and the high-dose group were significantly lower than those of the control group (p<0.01).

虽然已经在此示出并描述了本发明的优选实施方式，但是对本领域技术人员而言应该显而易见是这样的实施方式仅以举例方式提供。在不偏离本发明的情况下众多变化、改变和取代现在将被本领域的普通技术人员想到。应该理解的是，在实践本发明中可以采用在此描述的本发明的实施方式的不同替代方案。While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications and substitutions will now occur to those of ordinary skill in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

部分序列信息:

Partial sequence information:

Claims

RNA，其包括编码SARS-CoV-2的抗原性多肽或者其免疫原性片段或变体的开放阅读框，其中所述抗原性多肽选自SARS-CoV-2的受体结合结构域、S蛋白、其变体或者它们的免疫原性片段，优选地所述SARS-CoV-2是SARS-CoV-2 Delta变异病毒株。RNA, which includes an open reading frame encoding an antigenic polypeptide of SARS-CoV-2 or an immunogenic fragment or variant thereof, wherein the antigenic polypeptide is selected from the group consisting of the receptor binding domain and S protein of SARS-CoV-2. , variants thereof or immunogenic fragments thereof, preferably the SARS-CoV-2 is a SARS-CoV-2 Delta variant strain.
权利要求1所述的RNA，其中所述抗原性多肽或者其免疫原性片段或变体包含SARS-CoV-2多肽或其变体的一个或多个免疫原性表位；The RNA of claim 1, wherein the antigenic polypeptide or immunogenic fragment or variant thereof comprises one or more immunogenic epitopes of a SARS-CoV-2 polypeptide or variant thereof;

优选地，所述抗原性多肽或者其免疫原性片段或变体选自SARS-CoV-2 Delta变异病毒株的S蛋白，优选地是SARS-CoV-2 Delta变异病毒株的S蛋白变体，更优选地所述S蛋白变体选自Delta S-2P，其突变是K984P和V985P；以及Delta S-6P，其突变是F815P、A890P、A897P、A940P、K984P和V985P，Preferably, the antigenic polypeptide or immunogenic fragment or variant thereof is selected from the S protein of the SARS-CoV-2 Delta variant virus strain, preferably the S protein variant of the SARS-CoV-2 Delta variant virus strain, More preferably, the S protein variant is selected from Delta S-2P, whose mutations are K984P and V985P; and Delta S-6P, whose mutations are F815P, A890P, A897P, A940P, K984P and V985P,

优选地，所述抗原性多肽或者其免疫原性片段或变体包含SEQ ID NO：1、2或3的氨基酸17-1271的氨基酸序列，与SEQ ID NO：1、2或3的氨基酸17-1271的氨基酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的氨基酸序列；和/或Preferably, the antigenic polypeptide or immunogenic fragment or variant thereof comprises the amino acid sequence of amino acids 17-1271 of SEQ ID NO: 1, 2 or 3, and the amino acid sequence 17-1271 of SEQ ID NO: 1, 2 or 3. The amino acid sequence of 1271 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical; and/or

编码所述抗原性多肽或者其免疫原性片段或变体的所述RNA包含SEQ ID NO：4、5或6的核苷酸49-3813的核苷酸序列，与SEQ ID NO：4、5或6的核苷酸49-3813的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列。The RNA encoding the antigenic polypeptide or an immunogenic fragment or variant thereof includes the nucleotide sequence of nucleotides 49-3813 of SEQ ID NO: 4, 5 or 6, which is the same as SEQ ID NO: 4, 5 or the nucleotide sequence of nucleotides 49-3813 of 6 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
权利要求1或2所述的RNA，所述编码SARS-CoV-2的抗原性多肽或者其免疫原性片段或变体的开放阅读框还包含分泌信号肽，所述分泌信号肽优选地通过N-末端融合至所述的抗原性多肽或者其免疫原性片段或变体，所述分泌信号肽优选地是S蛋白的分泌信号肽， The RNA of claim 1 or 2, the open reading frame encoding the antigenic polypeptide of SARS-CoV-2 or an immunogenic fragment or variant thereof also contains a secretion signal peptide, preferably by N - terminally fused to the antigenic polypeptide or an immunogenic fragment or variant thereof, the secretion signal peptide is preferably the secretion signal peptide of the S protein,

优选地，所述分泌信号肽包含SEQ ID NO：1、2或3的氨基酸1-16的氨基酸序列，与SEQ ID NO：1、2或3的氨基酸1-16的氨基酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的氨基酸序列，或者SEQ ID NO：1、2或3的氨基酸1-16的氨基酸序列的功能片段或与SEQ ID NO：1、2或3的氨基酸1-16的氨基酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的氨基酸序列的功能片段；和/或Preferably, the secretion signal peptide comprises the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3, which is at least 99% identical to the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3. An amino acid sequence that is 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or a functional fragment of the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3 or is identical to SEQ ID NO: 1, 2 or 3. ID NO: A functional fragment with an amino acid sequence of at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of amino acids 1-16 of 1, 2 or 3; and / or

编码所述分泌信号肽的RNA包含SEQ ID NO：4、5或6的核苷酸1-48的核苷酸序列，与SEQ ID NO：4、5或6的核苷酸1-48的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列，或者SEQ ID NO：4、5或6的核苷酸1-48的核苷酸序列的片段或与SEQ ID NO：4、5或6的核苷酸1-48的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列的片段。The RNA encoding the secretion signal peptide includes the nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4, 5 or 6, and the nucleic acid sequence of nucleotides 1-48 of SEQ ID NO: 4, 5 or 6. A nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity, or the nucleotides of SEQ ID NO: 4, 5 or 6 A fragment of the nucleotide sequence 1-48 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, Fragments of nucleotide sequences that are 90%, 85% or 80% identical.
权利要求1或2所述的RNA，其中所述RNA为mRNA、环状RNA和自复制RNA，优选地所述RNA适合多肽的细胞内表达。The RNA of claim 1 or 2, wherein the RNA is mRNA, circular RNA and self-replicating RNA, preferably the RNA is suitable for intracellular expression of polypeptides.
权利要求1或2所述的RNA，其中所述RNA是修饰的RNA，其通过用修饰的尿苷残基取代一些或全部尿苷残基进行修饰，优选地所述修饰的尿苷是N1-甲基-假尿苷。The RNA of claim 1 or 2, wherein the RNA is a modified RNA modified by replacing some or all of the uridine residues with a modified uridine residue, preferably the modified uridine is N1- Methyl-pseudouridine.
权利要求1或2所述的RNA，其中所述RNA还包括一个或多个针对RNA在稳定性和翻译效率方面的最大效力进行优化的结构元件，优选地所述结构元件包括：5’帽、5’UTR、3’UTR和polyA尾序列。The RNA of claim 1 or 2, wherein the RNA also includes one or more structural elements optimized for the maximum effectiveness of the RNA in terms of stability and translation efficiency. Preferably, the structural elements include: 5' cap, 5'UTR, 3'UTR and polyA tail sequences.
权利要求6所述的RNA，其中所述5’帽是或包含cap1结构；更优选地，所述5’帽是m7G(5’)ppp(5’)(2’-OMeA)pG。 The RNA of claim 6, wherein the 5' cap is or comprises a cap1 structure; more preferably, the 5' cap is m7G(5')ppp(5')(2'-OMeA)pG.
权利要求6所述的RNA，其中所述5'-UTR是人β-珠蛋白mRNA的5'-UTR序列，任选地具有优化的Kozak序列；更优选地，所述5’UTR包含SEQ ID NO：7的核苷酸序列，或与SEQ ID NO：7的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列。The RNA of claim 6, wherein the 5'-UTR is the 5'-UTR sequence of human β-globin mRNA, optionally with an optimized Kozak sequence; more preferably, the 5'UTR comprises SEQ ID The nucleotide sequence of NO:7, or a nucleic acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO:7 nucleotide sequence.
权利要求6所述的RNA，其中所述3'-UTR是人β-珠蛋白mRNA的两个重复的3'-UTR；更优选地，所述3’UTR包含SEQ ID NO：8的核苷酸序列，或与SEQ ID NO：8的核苷酸序列具有至少99％、98％、97％、96％、95％、90％、85％或80％相同性的核苷酸序列。The RNA of claim 6, wherein the 3'-UTR is the two repeated 3'-UTR of human β-globin mRNA; more preferably, the 3'UTR comprises the nucleoside of SEQ ID NO: 8 acid sequence, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 8.
权利要求6所述的RNA，其中所述polyA尾序列包含至少50、至少60或至少100个A核苷酸；更优选地，所述polyA尾序列包含SEQ ID NO：9的核苷酸序列，或由SEQ ID NO：9的核苷酸序列组成。The RNA of claim 6, wherein the polyA tail sequence comprises at least 50, at least 60 or at least 100 A nucleotides; more preferably, the polyA tail sequence comprises the nucleotide sequence of SEQ ID NO: 9, Or consisting of the nucleotide sequence of SEQ ID NO: 9.
组合物，其包含权利要求1-10中任一项所述的RNA。A composition comprising the RNA of any one of claims 1-10.
权利要求11所述的组合物，其中所述组合物配制为或待配制为液体、固体或其组合，优选地所述组合物配制为或待配制为用于注射或其他给药方式，优选地所述组合物配制为或待配制为用于肌肉内注射。The composition of claim 11, wherein the composition is formulated or to be formulated as a liquid, solid or a combination thereof, preferably the composition is formulated or to be formulated for injection or other administration, preferably The composition is or is to be formulated for intramuscular injection.
权利要求11或12所述的组合物，其中所述RNA与蛋白和/或脂质复合，以产生用于施用的RNA-颗粒。The composition of claim 11 or 12, wherein the RNA is complexed with proteins and/or lipids to produce RNA-particles for administration.
权利要求13所述的组合物，其中将所述RNA配制于脂质纳米颗粒中，所述脂质纳米颗粒包含阳离子可电离的脂质、磷脂、胆固醇和聚乙二醇(PEG)-脂质；The composition of claim 13, wherein the RNA is formulated in lipid sodium In rice particles, the lipid nanoparticles comprise cationic ionizable lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids;

优选地所述脂质纳米颗粒包含((4-羟基丁基)氮烷二基)双(己烷-6，1-二基)双(2-癸酸己酯)、2-[(聚乙二醇)-2000]-N，N-双十四烷基乙酰胺、1，2-二硬脂酰基-sn-甘油-3-磷酸胆碱和胆固醇。Preferably, the lipid nanoparticles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene Diol)-2000]-N,N-distetradecyl acetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.
权利要求11所述的组合物，其中将所述RNA配制为或待配制为胶体；The composition of claim 11, wherein the RNA is formulated or to be formulated as a colloid;

优选地，所述RNA配制为颗粒，在形成的胶体分散相中存在50％或更多、75％或更多或者85％或更多的RNA；Preferably, the RNA is formulated as particles, and 50% or more, 75% or more or 85% or more RNA is present in the colloidal dispersed phase formed;

更优选地所述颗粒通过使溶于水相中的RNA暴露于溶于有机相中的脂质形成，其中优选地所述有机相包含乙醇；More preferably the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase, wherein preferably the organic phase comprises ethanol;

还优选地，所述颗粒通过使溶于水相中的RNA暴露于分散于水相中的脂质形成，其中优选地所述分散于水相中的脂质形成脂质体。It is also preferred that the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dispersed in the aqueous phase, wherein preferably the lipids dispersed in the aqueous phase form liposomes.
权利要求11或12所述的组合物，其中所述RNA以1μg-100μg每剂量的范围的量存在于所述组合物中。The composition of claim 11 or 12, wherein the RNA is present in the composition in an amount ranging from 1 μg to 100 μg per dose.
权利要求1-10中任一项所述RNA或权利要求11-16中任一项所述的组合物在制备药物中的用途，所述药物为疫苗，所述药物进一步包含一种或多种药学上可接受的载剂、稀释剂和/或赋形剂。The use of RNA according to any one of claims 1 to 10 or the composition according to any one of claims 11 to 16 in the preparation of medicines, said medicines being vaccines, said medicines further comprising one or more Pharmaceutically acceptable carriers, diluents and/or excipients.
权利要求17所述的用途，其中所述药物用于在受试者中诱导针对冠状病毒的免疫应答，优选地针对冠状病毒抗原的特异性免疫应答。The use of claim 17, wherein the medicament is used to induce an immune response against coronavirus in a subject, preferably a specific immune response against coronavirus antigens.
权利要求17所述的用途，其中所述药物用于冠状病毒感染的治疗或预防性治疗。 The use of claim 17, wherein the medicament is used for the treatment or preventive treatment of coronavirus infection.
权利要求17-19中任一项的用途，其中所述冠状病毒为β冠状病毒，优选地所述冠状病毒为沙贝病毒，更优选地所述冠状病毒为SARS-CoV-2，进一步优选地所述冠状病毒包括：新冠病毒原始毒株(GD108)、SARS-CoV-2 Alpha变异病毒株、SARS-CoV-2 Beta变异病毒株、SARS-CoV-2 Delta变异病毒株和SARS-CoV-2 Omicron变异病毒株。The use of any one of claims 17-19, wherein the coronavirus is a beta coronavirus, preferably the coronavirus is Sabei virus, more preferably the coronavirus is SARS-CoV-2, further preferably The coronavirus includes: the original strain of the new coronavirus (GD108), SARS-CoV-2 Alpha variant strain, SARS-CoV-2 Beta variant strain, SARS-CoV-2 Delta variant strain and SARS-CoV-2 Omicron mutant virus strains.
权利要求17-19中任一项的用途，其中所述受试者为哺乳动物，优选地所述受试者为小鼠或猴，还优选地所述受试者为人类。The use of any one of claims 17-19, wherein the subject is a mammal, preferably the subject is a mouse or a monkey, and further preferably the subject is a human.
权利要求1-10中任一项所述RNA、权利要求11-16中任一项所述的组合物或权利要求17-21中任一项所述的用途，其中当所述RNA、组合物或药物给予人源细胞时，实现可检测的所述抗原性多肽或者其免疫原性片段或变体表达，并且优选地这种表达持续至少24小时或更长的时间段。The RNA according to any one of claims 1 to 10, the composition according to any one of claims 11 to 16, or the use according to any one of claims 17 to 21, wherein when the RNA, the composition Or when the drug is administered to human cells, detectable expression of the antigenic polypeptide or immunogenic fragment or variant thereof is achieved, and preferably such expression lasts for at least 24 hours or longer.
权利要求1-10中任一项所述RNA、权利要求11-16中任一项所述的组合物或权利要求17-21中任一项所述的用途，其中施用所述RNA、组合物或药物在受试者中产生免疫效果，所述免疫效果包括产生SARS-CoV-2中和抗体和/或T细胞应答，特别是稳健的TH1型T细胞应答，优选地CD4+和/或CD8+T细胞应答。The RNA of any one of claims 1-10, the composition of any one of claims 11-16, or the use of any one of claims 17-21, wherein the RNA, composition or the drug produces an immune effect in the subject, said immune effect including the production of SARS-CoV-2 neutralizing antibodies and/or T cell responses, in particular robust TH1 type T cell responses, preferably CD4+ and/or CD8+ T cell response.
权利要求1-10中任一项所述RNA、权利要求11-16中任一项所述的组合物或权利要求17-21中任一项所述的用途，其中施用所述RNA、组合物或药物在受试者中产生免疫应答，The RNA of any one of claims 1-10, the composition of any one of claims 11-16, or the use of any one of claims 17-21, wherein the RNA, composition or the drug produces an immune response in the subject,

优选地，所述免疫应答包括产生针对SARS-CoV-2刺突蛋白的S1亚基的结合抗体滴度，更优选地所述免疫应答包括产生针对SARS-CoV-2病毒的中和抗体滴度。 Preferably, the immune response includes the generation of binding antibody titers directed against the S1 subunit of the SARS-CoV-2 spike protein, and more preferably the immune response includes the generation of neutralizing antibody titers against the SARS-CoV-2 virus. .
权利要求1-10中任一项所述RNA、权利要求11-16中任一项所述的组合物或权利要求17-21中任一项所述的用途，其中在将所述RNA、组合物或药物施用给受试者之后7天，受试者的血清显示针对所述开放阅读框编码的多肽的抗体的产生。The RNA of any one of claims 1-10, the composition of any one of claims 11-16 or the use of any one of claims 17-21, wherein the RNA, combined Seven days after administration of the substance or drug to the subject, the subject's serum shows the production of antibodies directed against the polypeptide encoded by the open reading frame.
权利要求1-10中任一项所述RNA、权利要求11-16中任一项所述的组合物或权利要求17-21中任一项所述的用途，其中在将所述RNA、组合物或药物施用给受试者之后14天，受试者的血清显示病毒中和活性。The RNA of any one of claims 1-10, the composition of any one of claims 11-16 or the use of any one of claims 17-21, wherein the RNA, combined The subject's serum showed virus-neutralizing activity 14 days after the substance or drug was administered to the subject.
权利要求23-26中任一项所述RNA、组合物或用途，其中所述受试者为哺乳动物，优选地所述受试者为小鼠，还优选地所述受试者为人类。The RNA, composition or use of any one of claims 23-26, wherein the subject is a mammal, preferably the subject is a mouse, and further preferably the subject is a human.
一种制备疫苗的方法，包括将权利要求1-10中任一项所述的RNA配制于脂质纳米颗粒中，所述脂质纳米颗粒包含阳离子可电离的脂质、磷脂、胆固醇和聚乙二醇(PEG)-脂质；A method for preparing a vaccine, comprising formulating the RNA according to any one of claims 1 to 10 into lipid nanoparticles, the lipid nanoparticles comprising cationic ionizable lipids, phospholipids, cholesterol and polyethylene glycol. Diol (PEG)-lipid;

优选地所述脂质纳米颗粒包含((4-羟基丁基)氮烷二基)双(己烷-6，1-二基)双(2-癸酸己酯)、2-[(聚乙二醇)-2000]-N，N-双十四烷基乙酰胺、1，2-二硬脂酰基-sn-甘油-3-磷酸胆碱和胆固醇。 Preferably, the lipid nanoparticles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene Diol)-2000]-N,N-distetradecyl acetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.