MRNA vaccine for preventing novel coronavirus olmitron mutant strain

Abstract

The invention relates to an mRNA (messenger ribonucleic acid) vaccine for preventing a novel coronavirus olmitron mutant strain. The mRNA vaccine comprises the following main components: mRNA with a mutation site and codon optimization, and lipid nanoparticles, the mRNA vaccine has a very good immune effect on a plurality of novel coronavirus Omeke virus strains.

Description

mRNA vaccine to prevent the novel coronavirus Omicron mutant

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to prior applications filed with the State Intellectual Property Office of China on May 30, 2023, with patent number 202310622591.5, entitled “Novel Coronavirus mRNA Vaccine for Preventing Omicron Mutants,” and with patent number 202410122989.7, entitled “Novel Coronavirus mRNA Vaccine for Preventing Omicron Mutants,” filed with the State Intellectual Property Office of China on January 29, 2024. The entire contents of both prior applications are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of biomedicine technology, and specifically relates to an mRNA vaccine against the novel coronavirus Omicron mutant strain and a preparation method thereof. Background Art

[0004] The new coronavirus (SARS-CoV-2, hereinafter referred to as the new coronavirus) is a type of coronavirus (CoVs) belonging to the Coronaviridae family of the order Nidovirales. It is widely present in nature. The virus particles are spherical or oval, with polymorphism, and a diameter of about 60 to 220 nm. The virus has an envelope with spikes (also known as S protein) on it. The internal genome is single-stranded positive-strand RNA (+ssRNA). The entire virus particle looks like a crown or corona under an electron microscope, hence the name. The new coronavirus genome is 29.9kb in size, consisting of 11 genomes, encoding 12 proteins, including spike protein (S protein), envelope protein, and capsid protein. Among them, the spike protein (S protein) is considered to be the most important viral surface membrane protein used by coronaviruses to invade cells. It contains two subunits, S1 and S2. The S1 subunit is responsible for binding to the host cell receptor ACE2, while the S2 subunit is responsible for anchoring the S protein to the host cell membrane and mediating the fusion of the viral envelope with the host cell membrane. The S1 subunit includes the N-Terminal Domain (NTD) and the Receptor Binding Domain (RBD), of which the RBD is involved in binding to the host cell receptor ACE2; the S2 subunit includes the Heptad Repeat (HR), the Central Helix (CH), and the Connector Domain (CD). In addition, there is a Furin cleavage site at the junction of the S1 and S2 subunits. Studies have shown that the S protein is considered the most likely vaccine antigen choice. Relying on the interaction between the S protein and human angiotensin-converting enzyme 2 (ACE2), the new coronavirus invades human respiratory epithelial cells and induces related diseases.

[0005] Since 2020, a variety of SARS-CoV-2 mutant strains have emerged and have begun to spread globally, including B.1.1.7 (British variant, Alpha strain, key mutations: D614G, N501Y, P681H), B.1.351 (South African variant, Beta strain, key mutations: E484K, N501Y, K417N), B.1.617.1 (first-generation Indian mutant, Kappa strain, key mutations: L452R, E484Q, D614G) and B.1.617.2 (second-generation Indian mutant, Delta strain, key mutations: L452R, E484Q, D614G), P.1 (Brazilian mutant, key mutations: K417T, E484K, N501Y), and the Omicron strain that appeared in late November 2021. As of 2023, the Omicron strains have become the predominant strain globally. The latest WHO report indicates that 77 countries and regions have reported cases of Omicron infection, suggesting that the strain has likely spread to most countries and regions worldwide. Furthermore, according to the latest data released by the China Centers for Disease Control and Prevention, from September 26, 2022, to April 27, 2023, a total of 43,777 valid genome sequences of local cases of COVID-19 were reported nationwide. These sequences were all Omicron variants (BA.1, BA.2, BA.5, BF.7, BQ.1, XBB, etc.), covering 174 evolutionary branches.

[0006] As third-generation vaccines, mRNA vaccines offer unique advantages over traditional vaccines. First, they are produced through an enzymatic in vitro transcription process, independent of cell amplification. This allows for easy monitoring and quality control of all production processes. Furthermore, they eliminate the need for cell culture, antigen extraction, and purification, significantly shortening production time. This allows for easy mass production and increases vaccine production capacity, easily reaching hundreds of millions or even billions of doses annually. This is crucial for rapidly responding to emerging infectious diseases worldwide. Second, mRNA vaccines can induce both humoral and cellular immunity, providing protection through multiple mechanisms and achieving higher efficacy. Currently, most vaccines target the original or delta strains of the novel coronavirus. The only mRNA vaccines targeting the Omicron strain are the Moderna and Pfizer / BioNTech bivalent COVID-19 vaccines, both approved for emergency use by the U.S. Food and Drug Administration (FDA), and their overall immune efficacy is limited. These bivalent COVID-19 vaccines target both the original strain and the Omicron BA.4 and BA.5 variants. Based on the prevalence of the above-mentioned Omicron strains and the progress of vaccine research and development, there is still a huge unmet market demand for iterative mRNA vaccine products targeting the new coronavirus Omicron strain.

[0007] Summary of the Invention

[0008] In response to the above defects of the prior art, the present invention provides an immune composition (e.g., an mRNA vaccine). The present invention designs an mRNA vaccine for the S protein coding region of each strain of SARS-CoV-2 Omicron virus, using in vitro transcribed mRNA. The key mutations in the S protein sequence of each strain of Omicron (BA.5, BA.2.75, BQ.1, BF.7, XBB, XBB.1.5, CH.1.1, XBB.1.16) are fully investigated, and an mRNA vaccine that is effective for each strain type is designed. In addition, in order to make the mRNA vaccine more suitable for expression in human cells, the present invention codon-optimizes the nucleic acid sequence of the mRNA vaccine and develops an mRNA vaccine that efficiently and stably translates and expresses the antigen S protein. The mRNA of the present invention is delivered into the body and translated into the S protein (antigen protein) of each strain type of Omicron, causing the body to produce humoral and cellular immunity, and produce memory B cells. When exposed to each strain type of Omicron virus, it can effectively prevent the infection of multiple mutants. Even a very small dose (e.g., 1.0 μg) can induce protection in animals against multiple mutants of SARS-CoV-2. It is significantly superior to existing vaccines in terms of safety and effectiveness, achieving unexpected technical results.

[0009] To achieve the above objectives, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a polynucleotide (e.g., mRNA) encoding at least one antigenic peptide or protein derived from SARS-CoV-2 coronavirus or an immunogenic fragment or immunogenic variant thereof, wherein the polynucleotide comprises at least one open reading frame connected to a heterologous untranslated region (UTR), and the polynucleotide has a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs. 1-19;

[0011] In some embodiments, the polynucleotide is suitable for use in the production of a vaccine;

[0012] In some embodiments, the at least one antigenic peptide or protein comprises or consists of at least one protein derived from a structural protein, an accessory protein, or a replication protein, or a fragment or immunogenic variant of any of the foregoing.

[0013] In some embodiments, the structural protein is or is derived from a spike protein (S protein), an envelope protein (E protein), a membrane protein (M protein) or a nucleocapsid protein (N protein) or an immunological fragment or variant thereof.

[0014] In some embodiments, the spike protein (S) is selected from the spike protein S1 protein or its immune fragment and variants thereof, S2 protein or its immune fragment and variants thereof.

[0015] In some embodiments, a polynucleotide of the invention may comprise or consist of a nucleotide sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 1-19.

[0016] In some embodiments, at least one coding region of a polynucleotide of the present invention encodes a protein containing mutations Spike_K986P and Spike_V987P, wherein the mutation sites are mutation sites relative to the S protein of the SARS-CoV-2 Wuhan-Hu-1 strain (the genome sequence is shown in NCBI sequence number: NC_045512.2), and the coding region comprises a nucleotide sequence optimized for G / C content. It should be understood that in this application, "Spike_" represents the amino acid number counted with the spike protein (Spike protein, or S protein) of the SARS-CoV-2 Wuhan-Hu-1 strain (the genome sequence is shown in NCBI sequence number: NC_045512.2, the sequence of the Wuhan-Hu-1 strain S protein is recorded under NCBI accession number YP_009724390.1) as the reference sequence. For example, Spike_K986P and Spike_V987P respectively indicate that position 986 relative to the reference sequence YP_009724390.1 is mutated from K to P, and position 987 relative to the reference sequence YP_009724390.1 is mutated from V to P.

[0017] In some embodiments, the heterologous untranslated region (UTR) includes at least one heterologous 5'UTR and / or 3'UTR, and the 5'UTR comprises a portion of a KOZAK sequence or consists of a KOZAK sequence with a start codon.

[0018] In some embodiments, the 3'UTR can also be selected from the 3'UTR of PSMB3, ALB7, α-globin, CASP1, COX6B1, GNAS, NDUFA1, DH143, gp130, hHBB, hHBA1, CYBA (cytochrome b-245alpha chain), rabbit β-globin, hepatitis B virus (HBV), VEEV (Venezuelan equine encephalitis virus) virus, rps9 (Ribosomal Protein S9), FIG4 (FIG4 Phosphoinositide 5-Phosphatase), human albumin hHBB (human hemoglobin subunit beta) or HBA1 (human Hemoglobin Subunit Alpha 1), or a homolog, fragment or variant from any of these genes.

[0019] In some embodiments, the 5'UTR is selected from the 5'UTR of African Xenopus or human α-globin or β-globin, human cytochrome b-245a polypeptide, hydroxysteroid (17b) dehydrogenase, and tobacco etch virus, alpha-1-globin, HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B or UBQLN2, or a homolog, fragment or variant from any of these genes.

[0020] In some embodiments, the nucleotide comprises at least one 3'-poly(A) sequence, preferably comprises 30 to 200 adenosine nucleotides and / or at least one 3'-poly(A) sequence.

[0021] In some embodiments, the mRNA further comprises a 5' guanosine cap selected from one or more of the following: m7Gppp (2'OMeA)pG、m7GpppApA、m7GpppApC、m7GpppApG、m7GpppApU、m7GpppCpA、m7GpppCpC、m7GpppCpG、m7GpppCpU、m7Gppp GpA、m7GpppGpG、m7GpppGpG、m7GpppGpU、m7GpppUpA、m7GpppUpC、m7GpppUpG、m7GpppUpU、m7Gpppm6ApG、m7G 3’Ome pppApA、m7G 3’Ome pppApC、m7G 3’Ome pppApU、m7G 3’Ome pppApG、m7G 3’Ome pppCpA、m7G 3’Ome pppCpC、m7G 3’Ome pppCpG、m7G 3’Ome pppCpU、m7G 3’Ome pppUpA、m7G 3’Ome pppUpC、m7G 3’Ome pppUpG、m7G 3’Ome pppUpU、m7G 3’Ome pppA 2’Ome pG、m7G 3’Ome pppA 2’Ome pC、m7G 3’Ome pppA 2’Ome pU、m7G 3’Ome pppA 2’Ome pA、m7G 3’Ome pppC 2’Ome pA、m7G 3’Ome pppC 2’Ome pU、m7G 3’Ome pppC 2’Ome pG、m7G 3’Ome pppC 2’Ome pC、m7G 3’Ome pppG 2’Ome pA、m7G 3’Ome pppG 2’Ome pU、m7G 3’Ome pppG 2’Ome pG、m7G 3’Ome pppG 2’Ome pC、m7G 3’Ome pppU 2’Ome pA、m7G 3’Ome pppU 2’Ome pU、m7G 3’Ome pppU 2’Ome pG、m7G3’Ome pppU 2’Ome pC.

[0022] In some embodiments, the polynucleotide comprises at least the following structure:

[0023] (a) 5'-cap structure; the 5'-cap structure is preferably any one of the following: m7G, cap0, cap1, cap2, modified cap0 or modified cap1 structure, preferably cap1 structure, most preferably m7G(5')ppp(5')(2'OMeA)pG (i.e., m7Gppp(2'OMeA)pG);

[0024] (b) a 3'-poly(A) sequence comprising 30 to 200 adenosine nucleotides and the 3'-terminal nucleotide is adenosine, preferably 50-200 A's, more preferably 80-200 A's;

[0025] (c) a 5'UTR of 10-200 nucleotides, preferably 15-150 nucleotides in length, preferably comprising pVAX and / or alpha-1-globin 5'UTR, KOZAK sequence, and / or TEV sequence;

[0026] (d) 3'UTR, preferably comprising one or more sequences selected from the group consisting of human gp130 3'UTR, DH143 3'UTR, hHBB 3'UTR, and / or hHBA1 3'UTR sequences.

[0027] In some embodiments, the 3'-poly(A) comprises the nucleotide sequence of SEQ ID NO.24.

[0028] In some embodiments, the 5'UTR comprises a nucleotide sequence selected from any one or more of SEQ ID NOs. 21-23;

[0029] In some embodiments, the 5'UTR is composed of the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22 in tandem from 5' to 3'.

[0030] In some embodiments, the 3'UTR comprises a nucleotide sequence selected from any one or more of SEQ ID NOs. 25-28;

[0031] In some embodiments, the 3'UTR is composed of the sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26 in series from 5' to 3'.

[0032] In some embodiments, the mRNA comprises chemically modified bases or analogs, including 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, 1-methyl pseudouridine (N1-Methyl-Pseudo-UTP), and pseudouridine; preferably, 1-methyl pseudouridine partially replaces uridine; and most preferably, 1-methyl pseudouridine completely replaces uracil, so that each uracil in the sequence is replaced by 1-methyl pseudouridine.

[0033] In some embodiments, the aforementioned polynucleotide is DNA or RNA.

[0034] In some embodiments, the aforementioned polynucleotide encodes RNA.

[0035] In some embodiments, the coding RNA is mRNA, self-replicating RNA, circular RNA, or replicon RNA, preferably mRNA.

[0036] In some embodiments, the present invention relates to an mRNA encoding a coronavirus antigen comprising amino acids that are at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or at least 99% or 100% identical to the amino acid sequence of any one or more of SEQ ID NOs. 30-37.

[0037] In some embodiments, the aforementioned spike protein (S) may comprise or consist of an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one or more of SEQ ID NO.30-37.

[0038] In some embodiments, the mRNA encodes a coronavirus antigen comprising at least one mutation relative to the S protein of the wild strain (NCBI sequence number: NC_045512.2): Spike_T19I, Spike_L24del, Spike_P25del, Spike_P26del, Spike_A27S, Spike_H69del, Spike_V70del, Spike_V83A, Spike_G142D, Spike_H146Q, Spike_W152R, Spike_F157L, Spike_E180V, Spike_Q183E, Spike_I210V, Spike_V213G, Spike_G252V, Spike_G257S, Spike_G339D, Spike_L368I, Spike_S371F, S pike_S373P, Spike_S375F, Spike_T376A, Spike_D405N, Spike_R408S, Spike_K417N, Spike_N44 0K, Spike_K444T, Spike_V445P, Spike_L452R, Spike_N460K, Spike_S477N, Spike_T478K, Spike_ E484A, Spike_F486V, Spike_F490S, Spike_Q498R, Spike_N501Y, Spike_Y505H, Spike_D614G, Spi ke_H655Y, Spike_N679K, Spike_P681H, Spike_N764K, Spike_D796Y, Spike_Q954H, Spike_N969K.

[0039] In a second aspect, the present invention provides a composition, preferably an immunogenic composition comprising at least one polynucleotide of the first aspect; suitably, the composition may comprise at least one polynucleotide, such as at least one encoding RNA, which is complexed with one or more lipids, encapsulated in one or more lipids or associated with one or more lipids to form lipid nanoparticles.

[0040] In some embodiments, the composition relates to a novel coronavirus nucleic acid vaccine, wherein the vaccine carrier is a lipid nanoparticle (LNP) comprising an ionizable cationic lipid, a structural lipid, a helper lipid, and a surfactant. In some embodiments, the ionizable cationic lipid is selected from SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200, DlinDMA, preferably SM-102, and the structure of SM-102 is as follows:

[0041] In some embodiments, the structured lipid is selected from cholesterol, and cholesterol derivatives, preferably cholesterol;

[0042] In some embodiments, the helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DSPC;

[0043] In some embodiments, the surfactant is selected from PEG2000-DMG, PEG-DSPE, DTDA-PEG2000, TPGS, preferably PEG2000-DMG.

[0044] In some embodiments, the lipid nanoparticles comprise 20-60 mol% ionizable cationic lipids, 25-55 mol% structural lipids, 5-25 mol% helper lipids, and 0.5-15 mol% surfactants, based on a molar percentage (mol%). The lipid nanoparticles comprise 20-50 mol% ionizable cationic lipids. For example, the lipid nanoparticles may comprise 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mol% ionizable cationic lipids.

[0045] In other embodiments, the lipid nanoparticles comprise 50-60 mol% ionizable cationic lipids. For example, the lipid nanoparticles may comprise 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 mol% ionizable cationic lipids.

[0046] In some embodiments, the lipid nanoparticle comprises 5-25 mol% DSPC, preferably 2-15 mol% DSPC; for example, the lipid nanoparticle may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mol% DSPC.

[0047] In some embodiments, the lipid nanoparticles comprise 25-55 mol% cholesterol, preferably 30-40 mol% cholesterol. For example, the lipid nanoparticles may comprise 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 mol% cholesterol.

[0048] In some embodiments, the lipid nanoparticles comprise 0.5-15 mol% DMG-PEG, preferably 1-2 mol% DMG-PEG. For example, the lipid nanoparticles may comprise 1, 1.5 or 2 mol% DMG-PEG.

[0049] In some embodiments, the lipid nanoparticles comprise 50 mol% ionizable cationic lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.

[0050] In some embodiments, the lipid nanoparticles comprise 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.

[0051] In some embodiments, the molar ratio of protonable nitrogen elements to phosphate groups of mRNA (N:P ratio) in the lipid nanoparticles of the present invention is about 2:1 to about 30:1.

[0052] In some embodiments, the molar ratio of protonable nitrogen elements to phosphate groups of mRNA (N:P ratio) in the lipid nanoparticles of the present invention is about 6:1.

[0053] In some embodiments, the molar ratio of protonable nitrogen elements to phosphate groups of mRNA (N:P ratio) in the lipid nanoparticles of the present invention is about 3:1.

[0054] In some embodiments, the lipid nanoparticles of the invention comprise a wt / wt ratio of the ionizable cationic lipid component to the RNA of about 10:1 to about 100:1.

[0055] In some embodiments, the lipid nanoparticles of the invention comprise a wt / wt ratio of ionizable cationic lipid component to RNA of about 20:1.

[0056] In some embodiments, the lipid nanoparticles of the invention comprise a wt / wt ratio of ionizable cationic lipid component to RNA of about 10:1.

[0057] In some embodiments, the lipid nanoparticles of the invention have an average diameter of from about 50 nm to about 150 nm.

[0058] In some embodiments, the lipid nanoparticles of the present invention have an average diameter of about 70 nm to about 120 nm, preferably 100-120 nm, and most preferably 100 nm.

[0059] In some embodiments, the novel coronavirus nucleic acid vaccine of the present invention further comprises: a buffer component and a cryoprotectant;

[0060] In some embodiments, the buffer can be selected from: citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium gluconate, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydrogen phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, dibasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, magnesium hydroxide, aluminum hydroxide, alginic acid, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, lauryl magnesium sulfate, sodium lauryl sulfate, and combinations thereof.

[0061] In some embodiments, the cryoprotectant can be selected from substances such as sugars / polyols, polymers, surfactants, amino acids and salts, wherein the sugar can be selected from lactose, sucrose, trehalose, galactose and the like.

[0062] In some embodiments, the amount of the cryoprotectant is 1 to 50% w / w, such as from 2 to 50% w / w, or from 4 to 45% w / w, or from 6 to 12% w / w, or preferably from 6 to 10% w / w, or most preferably from 7 to 9% w / w.

[0063] In some embodiments, the composition of the present invention includes the aforementioned lipid nanoparticles and an external phase (also referred to herein as "aqueous phase" or "external aqueous phase") buffer.

[0064] In some embodiments, the aqueous buffer comprises: tromethamine, sodium acetate, sucrose, pH=7-8.

[0065] In some embodiments, the content of tromethamine is selected from 10-30mmol / L, preferably 15-25mmol / L, preferably 15mmol / L, 15.5mmol / L, 16mmol / L, 16.5mmol / L, 17mmol / L, 17.5mmol / L, 18mmol / L, 18.5mmol / L, 19mmol / L, 19.5mmol / L, 20mmol / L, 20.5mmol / L, 21mmol / L, 21.5mmol / L, 22mmol / L, 22.5mmol / L, 23mmol / L, 23.5mmol / L, 24mmol / L, 24.5mmol / L, 25mmol / L, and most preferably 20mmol / L.

[0066] In some embodiments, the content of sodium acetate is selected from 0-20mmol / L, preferably 5-11mmol / L, preferably 5mmol / L, 5.5mmol / L, 6mmol / L, 6.5mmol / L, 7mmol / L, 7.5mmol / L, 8mmol / L, 8.5mmol / L, 9mmol / L, 9.5mmol / L, 10mmol / L, 10.5mmol / L, 10.6mmol / L, 10.7mmol / L, 10.8mmol / L, 10.9mmol / L, 11mmol / L, 11.5mmol / L, 12mmol / L, 12.5mmol / L, 13mmol / L, and most preferably 10.7mmol / L.

[0067] In some embodiments, the sucrose content (w / v) is selected from: 5-15%, preferably 7.5-10%, more preferably 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 9%, 9.5%, 10%, and most preferably 8.7%. It should be understood that all references to the percentage of sucrose content in this application refer to its concentration, i.e., the concentration (w / v) of sucrose in the aqueous buffer.

[0068] In some embodiments, the composition of the present invention includes at least one mRNA having a sequence as shown in any one of SEQ ID NO.1-19, lipid nanoparticles, 20 mmol / L tromethamine, 10.7 mmol / L sodium acetate, 8.7% sucrose, pH 7.0-8.0, wherein the lipid nanoparticles contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% PEG2000-DMG, N:P=6.

[0069] In a third aspect, the present invention provides a novel coronavirus vaccine comprising antigenic polypeptides of SARS-CoV-2.

[0070] In some embodiments, the antigenic polypeptide comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in any one or more of SEQ ID NOs.: 29-37.

[0071] In some embodiments, the antigen comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in any one or more of SEQ ID NOs.: 30-37.

[0072] In some embodiments, relative to the amino acid sequence of the spike protein of the novel coronavirus strain (NCBI sequence number: NC_045512.2), the antigen protein of the present application includes one or more of the following mutations: Spike_T19I, Spike_L24del, Spike_P25del, Spike_P26del, Spike_A27S, Spike_H69del, Spike_V70del, Spike_V83A, Spike_G142D, Spike_H146Q, Spike_W152R, Spike_F157L, Spike_E180V, Spike_Q183E, Spike_I210V, Spike_V213G, Spike_G252V, Spike_G257S, Spike_G339D, Spike_L368I, Spike_S371F, Spike_S373P, Spike_S375F,Spike_T376A,Spike_D405N,Spike_R408S,Spike_K417N,Spike_N440K,Spike_K444T, Spike_V445P,Spike_L452R,Spike_N460K,Spike_S477N,Spike_T478K,Spike_E484A,Spike_F486V, Spike_F490S,Spike_Q498R,Spike_N501Y,Spike_Y505H,Spike_D614G,Spike_H655Y,Spike_N679K, Spike_P681H, Spike_N764K, Spike_D796Y, Spike_Q954H, Spike_N969K, Spike_K986P, Spike_V987P.

[0073] In a fourth aspect, the present invention provides a novel coronavirus vaccine, preferably a SARS-CoV-2 vaccine, wherein the vaccine comprises at least one polynucleotide of the first aspect, or the composition of the second aspect, or at least one antigenic polypeptide of the third aspect.

[0074] In some embodiments, the vaccine is administered by intravenous injection, intramuscular injection or subcutaneous injection, preferably intramuscular injection.

[0075] In some embodiments, the dosage form of the vaccine can be selected from lyophilized powder injection, liquid injection, and inhalation preparation.

[0076] In a fifth aspect, the present invention provides a kit or kit of parts comprising at least one polynucleotide of the first aspect, and / or at least one composition of the second aspect, and / or at least one antigenic polypeptide of the third aspect, and / or at least one vaccine of the fourth aspect.

[0077] In a sixth aspect, the present invention provides a pharmaceutical composition comprising at least two separated components, wherein the at least two separated components are selected from the two nucleotides of the first aspect, and / or at least two compositions of the second aspect, and / or at least two antigenic polypeptides of the third aspect, and / or at least two vaccines of the fourth aspect.

[0078] In a seventh aspect, the present invention provides a method for treating or preventing coronavirus infection, preferably SARS-CoV-2 infection, in a subject, comprising administering to the subject at least one polynucleotide of the first aspect, and / or at least one composition of the second aspect, and / or at least one antigenic polypeptide of the third aspect, and / or at least one vaccine of the fourth aspect, which can effectively induce a neutralizing antibody response against SARS-CoV-2 in the subject.

[0079] In the eighth aspect, the present invention relates to a method for preparing a new coronavirus vaccine, comprising mixing a vaccine vector and the mRNA of the first aspect to obtain a new coronavirus vaccine.

[0080] In some embodiments, the vaccine carrier is a cationic lipid nanoparticle, and the preparation method specifically comprises the following steps:

[0081] (1) dissolving an ionizable cationic lipid, a structural lipid, an auxiliary lipid, and a surfactant in an organic solution according to a formula ratio to obtain an organic phase;

[0082] (2) dissolving the optimized mRNA in citrate buffer or sodium acetate solution to obtain an aqueous phase;

[0083] (3) mixing the organic phase of step (1) and the aqueous phase of step (2) to generate a mixed solution to obtain a novel coronavirus vaccine;

[0084] In some embodiments, the organic solution comprises anhydrous ethanol;

[0085] In some embodiments, the total concentration of the ionizable cationic lipid, structural lipid, helper lipid, and surfactant in the organic phase is 10-15 mg / ml;

[0086] In some embodiments, the concentration of mRNA in the aqueous phase is 0.01-1 mg / ml, preferably 0.1-0.2 mg / ml;

[0087] In some embodiments, the volume ratio of the organic phase to the aqueous phase is 1:2-4;

[0088] In some embodiments, the mixing is performed using a microfluidic device with a flow rate controlled to be ≥12 ml / min.

[0089] In the ninth aspect, the present application also relates to the use of the polynucleotide of the first aspect, and / or the composition of the second aspect, and / or the antigenic polypeptide of the third aspect, and / or the vaccine of the fourth aspect in preparing a vaccine.

[0090] In some embodiments, the vaccines include combination vaccines and multivalent vaccines.

[0091] Preferably, the vaccine is a SARS-CoV-2 virus mRNA vaccine, preferably the vaccine is a SARS-CoV-2 virus mutant vaccine, and the mutant strains are preferably B.1.1.7 (British mutant, Alpha strain, key mutations: D614G, N501Y, P681H), B.1.351 (South African mutant, Beta strain, key mutations: E484K, N501Y, K417N), B.1.617.1 (first-generation Indian mutant, Kappa strain, key mutations: L452R, E484Q, D614G) and B.1.617.2 (second-generation Indian mutant, Delta strain, key mutations: L452R, E484Q, D614G), P.1 (Brazilian mutant, key mutations: K417T, E484K, N501Y), and Omicron series strains (BA.5, BA.2.75, BQ.1, BF.7, XBB, XBB.1.5, CH.1.1, XBB.1.16), which contain at least one of the following mutations: Spike_T19I, Spike_L24del, Spike_P25del, Spike_P26del, Spike_A27S, Spike_H69del, Spike_V70del, Spike_V83A, Spike_G14 2D,Spike_H146Q,Spike_W152R,Spike_F157L,Spike_E180V,Spike_Q183E,Spike_I210V,Spike_V213G,Spike_G252V,Spike_G257S,Spike_ G339D,Spike_L368I,Spike_S371F,Spike_S373P,Spike_S375F,Spike_T376A,Spike_D405N,Spike_R408S,Spike_K417N,Spike_N440K,Spik e_K444T,Spike_V445P,Spike_L452R,Spike_N460K,Spike_S477N,Spike_T478K,Spike_E484A,Spike_F486V,Spike_F490S,Spike_Q498R,Sp ike_N501Y, Spike_Y505H, Spike_D614G, Spike_H655Y, Spike_N679K, Spike_P681H, Spike_N764K, Spike_D796Y, Spike_Q954H, Spike_N969K.

[0092] In the tenth aspect, the present invention provides a combination vaccine comprising a first vaccine and a second vaccine used simultaneously or sequentially, wherein the first vaccine is selected from the polynucleotide of the first aspect, and / or the composition of the second aspect, and / or the pharmaceutical composition of the sixth aspect.

[0093] In some embodiments, the second vaccine is selected from: attenuated or inactivated vaccines, adenovirus vaccines, mRNA vaccines, DNA vaccines, and recombinant protein vaccines.

[0094] In some embodiments, the second vaccine is selected from: Moderna (mRNA-1273), CureVac (CVnCoV), Johnson & Johnson (COVID-19 Vaccine Janssen), AstraZeneca (Vaxzevria), Pfizer / BioNTech (Comirnaty), Sputnik (Gam-COVID-Vac), Sinovac (COVID-19 Vaccine (Vero Cell), Novavax (NVX-CoV2373), Shijiazhuang Pharmaceutical Group's new crown mRNA vaccine SYS6006, CanSino's new crown vaccine, Beijing Sinopharm's new crown vaccine, and Chinese Academy of Sciences-Zhifei's recombinant new crown virus vaccine.

[0095] In some embodiments, the mRNA of the first vaccine and the second vaccine comprises a nucleotide sequence as shown in any one or more of SEQ ID NOs: 1-19, or the nucleotide sequence of the mRNA of the first vaccine and the second vaccine is as shown in the nucleotide sequence as shown in any one or more of SEQ ID NOs: 1-19.

[0096] In some embodiments, the vaccine described in the present application is suitable for sequential vaccination with one or more vaccines selected from the following groups, and the vaccine can be based on vaccines of any technical route, including but not limited to attenuated or inactivated vaccines, adenovirus vaccines, mRNA vaccines, DNA vaccines, recombinant protein vaccines, etc.

[0097] In some embodiments, the one or more vaccines are selected from: Moderna (mRNA-1273), CureVac (CVnCoV), Johnson & Johnson (COVID-19 Vaccine Janssen), AstraZeneca (Vaxzevria), Pfizer / BioNTech (Comirnaty), Sputnik (Gam-COVID-Vac), Sinovac (COVID-19 Vaccine (Vero Cell), Novavax (NVX-CoV2373), Shijiazhuang Pharmaceutical Group's new crown mRNA vaccine SYS6006, CanSino's new crown vaccine, Beijing Sinopharm's new crown vaccine, and Chinese Academy of Sciences-Zhifei's recombinant new crown virus vaccine.

[0098] In some embodiments, the mRNA of one or more vaccines can be mixed into one LNP for delivery, or the above mRNA can be mixed into different LNPs, and then the LNPs are mixed in a certain ratio before administration.

[0099] In some embodiments, in one relatively complete vaccination, the number of vaccinations of the one or more vaccines required to complete immunization can be 1 time, 2 times, 3 times or 4 times, and the interval between each vaccination can be 21 days, 28 days, 35 days, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0100] In some embodiments, the dosage of the vaccine is selected from 5μg-50μg, for example: 5μg, 10μg, 15μg, 20μg, 25μg, 30μg, 35μg, 40μg, 45μg, 50μg; preferably 10-30μg; most preferably 10μg, 20μg, 30μg.

[0101] In some embodiments, the vaccine of the present invention can be used as a booster shot for subjects who have already received two doses of an inactivated vaccine;

[0102] In an eleventh aspect, the present invention provides a novel coronavirus vaccine delivered by lipid nanoparticles, wherein the novel coronavirus vaccine contains lipid nanoparticles, optionally in one of the following schemes:

[0103] 1) The first reading frame and the second reading frame are respectively present in different nucleic acid molecules, and the nucleic acid molecule containing the first reading frame and the nucleic acid molecule containing the second reading frame are respectively encapsulated in different LNPs, that is, the new coronavirus vaccine comprises: LNPs encapsulated with a nucleic acid molecule containing the first open reading frame, and LNPs encapsulated with a nucleic acid molecule containing the second open reading frame;

[0104] 2) The first reading frame and the second reading frame are respectively present in different nucleic acid molecules, and the nucleic acid molecule containing the first reading frame and the nucleic acid molecule containing the second reading frame are mixed and encapsulated in the same LNP, that is, the new coronavirus vaccine comprises: LNPs encapsulated with a nucleic acid molecule containing the first open reading frame and a nucleic acid molecule containing the second open reading frame;

[0105] 3) The first reading frame and the second reading frame exist in the same nucleic acid molecule, which is encapsulated in LNP, that is, the new coronavirus vaccine comprises: LNP encapsulated with a nucleic acid molecule containing both the first open reading frame and the second open reading frame, wherein the first reading frame and the second reading frame represent different structural units capable of expressing the S protein.

[0106] In some embodiments, the vaccine is preferably delivered by lipid nanoparticles, and the novel coronavirus vaccine contains lipid nanoparticles, which can optionally be one of the following:

[0107] 1) The first reading frame encoding the antigen protein and the second reading frame encoding the antigen protein are respectively present in different mRNAs, and the mRNA molecules containing the first reading frame and the mRNA molecules containing the second reading frame are respectively encapsulated in different lipid nanoparticles;

[0108] 2) The first reading frame encoding the antigen protein and the second reading frame encoding the antigen protein are respectively present in different mRNA molecules, and the mRNA molecules containing the first reading frame and the mRNA molecules containing the second reading frame are mixed in a certain ratio and then encapsulated in the same lipid nanoparticle;

[0109] The mRNA molecule is selected from the nucleotide sequence shown in any one of SEQ ID NOs: 1-19, and the lipid nanoparticles contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG.

[0110] In some embodiments, LNPs containing nucleic acid molecules expressing reading frames of different strains are mixed in a ratio of 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, or 4:1 and then administered.

[0111] In some embodiments, nucleic acid molecules containing reading frames expressing different strains are mixed in a ratio of 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, or 4:1 and then encapsulated in the same LNP particle.

[0112] In some embodiments, the nucleic acid molecules expressing reading frames of different strains include mRNA of the first reading frame encoding the antigen protein and mRNA of the second reading frame mixed in the above ratio, and an adjustment of ±10% in the above ratio is still within the scope of protection of the present invention.

[0113] In some embodiments, the nucleic acid molecules expressing reading frames of different strains include mRNA encoding an antigenic protein in a first reading frame, an mRNA in a second reading frame, and an mRNA in a third reading frame, and the mRNAs are mixed in a ratio of 1:1:1, 1:1:2, 1:2:1, or 2:1:1 and then encapsulated in the same LNP particle. Adjustments of ±10% in the above ratios are still within the scope of protection of the present invention.

[0114] Beneficial effects of the present invention:

[0115] 1. The present invention fully examines the key mutations in the S protein sequence of each Omicron strain (BA.5, BA.2.75, BQ.1, BF.7, XBB, XBB.1.5, CH.1.1, XBB.1.16) and designs an mRNA vaccine that is effective against each strain.

[0116] 2. In order to achieve the best expression effect and stability of the mRNA sequence in vivo, the present invention optimizes the nucleotide coding sequence of the S protein coding region of each strain of SARS-CoV-2 Omicron virus, including: adjusting the codon preference for expression in the human body, adjusting the frequency of use of commonly used codons, increasing the GC content of the sequence, etc., and screening the optimized nucleotide sequence to finally determine the optimal sequence. The nucleotide sequence obtained in this application makes the transcribed mRNA structure more stable, and the target protein translation efficiency in mammals and humans is higher, which can effectively produce neutralizing antibodies, and induce the body to produce humoral and cellular immunity, and produce memory B cells and T cells; when exposed to various strains of Omicron virus, it can effectively prevent the infection of multiple mutants. Using a very small dose (for example, 1.0 μg) can induce animals to produce protective effects against multiple mutants of SARS-CoV-2.

[0117] 3. The cross-protection effect of immune serum from the bivalent SARS-CoV-2 mRNA vaccine (mRNA mixture 1) was superior to that of SYS6006 against the major circulating Omicron strains (BA.5, BQ.1, XBB.1.5, XBB.1.16, and the major circulating strain EG.5). The mRNAs of XBB.1.5 and BQ.1 showed a synergistic effect in cross-protection against the virus.

[0118] 4. The present invention uses LNP to deliver mRNA. mRNA has good stability in LNP, and the vaccine storage conditions are moderate. It can be stored stably for a long time at -20°C or higher temperature conditions (2-8°C). The transportation is relatively convenient and does not require high equipment. It is more suitable for economically underdeveloped or underdeveloped areas that are most in need of vaccination. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1: Bioanalyzer analysis of the purity and size of S protein mRNA from multiple Omicron mutants.

[0120] Note: The relevant strains and sequences are as follows:

[0121] BA.5(SEQ ID NO.5),BF.7 02(SEQ ID NO.13),BA.5(SEQ ID NO.4),BQ.1-02(SEQ ID NO.11),XBB.1.5(SEQ ID NO.15),BF.7-01(SEQ ID NO.12),XBB-01(SEQ ID NO.6),XBB-02(SEQ ID NO.7), BQ.1-1 (SEQ ID NO.10).

[0122] Figure 2: Immunoblotting and ELISA experiments verified the expression of Omicron BA.5-1 (SEQ ID NO. 4) and BA.5-2 (SEQ ID NO. 5) S proteins in host cells.

[0123] Figure 2A: Western blot detection; Figure 2B: ELISA detection.

[0124] Figure 3: Immunoblotting and ELISA experiments verified the expression of Omicron BF.7 mRNA-1 (SEQ ID NO.12) and BF.7 mRNA-2 (SEQ ID NO.13) S protein in host cells.

[0125] Figure 3A: Western blot detection; Figure 3B: ELISA detection.

[0126] Figure 4: Immunoblotting and ELISA experiments verified the expression of Omicron BQ.1-1 (SEQ ID NO.10) and BQ.1-2 (SEQ ID NO.11) S proteins in host cells.

[0127] Figure 4A: Western blot detection; Figure 4B: ELISA detection.

[0128] Figure 5: Immunoblotting and ELISA experiments verified the expression of Omicron XBB-1 (SEQ ID NO.6) and XBB-2 (SEQ ID NO.7) S proteins in host cells.

[0129] Figure 5A: Western blot detection Figure 5B: ELISA detection.

[0130] Figure 6: Immunoblotting and ELISA experiments verified the expression of Omicron XBB.1.5 (SEQ ID NO.15) S protein in host cells.

[0131] Figure 6A: Western blot detection Figure 6B: ELISA detection.

[0132] Figure 7: ELISA experiment verifies the expression of Omicron XBB.1.16 (SEQ ID NO.16) S protein in host cells.

[0133] Figure 8: Experiment on the binding of serum antibodies from mice immunized with Omicron BA.5-1 (SEQ ID NO.4) to S protein and neutralization of pseudovirus.

[0134] FIG8A shows antibody binding assay results; FIG8B shows pseudovirus neutralization assay results.

[0135] Figure 9: Experiments showing that serum antibodies from mice immunized with Omicron BF.7 mRNA-1 (SEQ ID NO.12) and BF.7 mRNA-2 (SEQ ID NO.13) bind to S protein and neutralize pseudovirus.

[0136] FIG9A shows antibody binding assay results; FIG9B shows pseudovirus neutralization assay results.

[0137] Figure 10: Experiments showing that serum antibodies from mice immunized with Omicron BQ.1-1 (SEQ ID NO.10) and BQ.1-2 (SEQ ID NO.11) bind to S protein and neutralize pseudovirus.

[0138] FIG10A shows antibody binding assay results; FIG10B shows pseudovirus neutralization assay results.

[0139] Figure 11: Experiments showing that serum antibodies from mice immunized with Omicron XBB-1 (SEQ ID NO. 6) and XBB-2 (SEQ ID NO. 7) bind to S protein and neutralize pseudovirus.

[0140] FIG11A shows antibody binding assay results; FIG11B shows pseudovirus neutralization assay results.

[0141] Figure 12 shows the pseudovirus neutralization experiment of sera from mice immunized with Omicron XBB-1 (SEQ ID NO. 6) and BQ.1-1 (SEQ ID NO. 10).

[0142] Figure 13 shows pseudovirus-neutralizing antibody activity in sera from mice injected with different S protein mRNA LNP formulations. Mice were immunized on days 0 and 14. The columns corresponding to each mRNA LNP formulation, from left to right, indicate the 50% inhibition dilution factor against the XBB, BQ.1, and XBB.1.5 strains, respectively.

[0143] Figure 14 shows the real virus neutralizing antibody activity of sera from mice injected with different S protein mRNA LNP formulations. Mice were immunized on days 0 and 14. The bars corresponding to each mRNA LNP formulation, from left to right, indicate the 50% inhibition dilution factor against BA.5, BF.7, XBB, and BQ.1 strains.

[0144] Figure 15: Changes in serum specific binding antibodies in mice injected with different S protein mRNA LNP formulations. Mice were immunized on days 0 and 21. BQ.1SP represents the binding antibody titer to the BQ.1 S protein, and XBB.1.5SP represents the binding antibody titer to the XBB.1.5 S protein.

[0145] Figure 16 shows the real virus neutralizing antibody activity of sera from mice injected with different S protein mRNA LNP formulations. Mice were immunized on days 0 and 21. The bars corresponding to each mRNA LNP formulation, from left to right, indicate the 50% inhibition dilution factor for BA.5, BQ.1, XBB.1.5, XBB.1.16, XBB.1.19, and EG.5, respectively.

[0146] Figure 17 shows pseudovirus-neutralizing antibody activity in sera from mice injected with different S protein mRNA LNP formulations. Mice were immunized on days 0 and 21. The columns for each mRNA LNP formulation, from left to right, indicate the 50% inhibition dilution factor for BA.4 / 5, BQ.1, XBB.1.5, XBB.1.16, and EG.5. DETAILED DESCRIPTION

[0147] Definition and explanation:

[0148] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0149] Although the numerical ranges and parameters shown in the broad scope of this application are approximate, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is inherently bound to contain a certain error (for example: ± 10%), which is caused by the standard deviation existing in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all subranges contained therein. For example, a range of "1 to 10" should be considered to include any and all subranges between a minimum of 1 and a maximum of 10 (including endpoints); that is, all subranges starting with a minimum of 1 or greater, such as 1 to 6.1, and subranges ending with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0150] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. It will also be understood that the skilled artisan can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleic acid molecule to reflect the codon usage of any particular host organism in which the polypeptide is expressed. Therefore, unless otherwise indicated, "polynucleotides encoding the protein or immunogenic fragment of the present application" include all polynucleotide sequences that are degenerate to each other and encode the same amino acid sequence.

[0151] antigen

[0152] Antigens as used herein are proteins that can induce an immune response (e.g., cause the immune system to produce antibodies against an antigen). In this article, unless otherwise indicated, the use of the term "antigen" includes immunogenic proteins and immunogenic fragments (immunogenic fragments that induce or can induce an immune response to at least one coronavirus). It should be understood that the term "protein" includes peptides and the term "antigen" includes antigenic fragments. Other molecules may also be antigenic, such as bacterial polysaccharides or a combination of protein and polysaccharide structures. The viral vaccine antigens described herein include viral proteins, viral protein fragments, and proteins designed and / or mutated from coronavirus SARS-CoV-2.

[0153] In addition, the term "antigen" as used herein will be recognized and understood by those of ordinary skill in the art, meaning a substance that can be recognized by the immune system, preferably by the adaptive immune system, and that can trigger an antigen-specific immune response, for example. By forming antibodies and / or antigen-specific T cells as part of an adaptive immune response. An antigen can be or can include a peptide or protein that can be presented to T cells by MHC. Also included are fragments, variants, and derivatives derived from, for example, a peptide or protein. The spike protein (S) of a coronavirus comprising at least one epitope, preferably a spike protein from SARS-CoV-2 (nCoV-2019) or a variant thereof, is designed as an antigen.

[0154] Antigenic peptide or protein: The term "antigenic peptide or protein" or "immunogenic peptide or protein" will be recognized and understood by those of ordinary skill in the art; it means a peptide, protein derived from a (antigenic or immunogenic) protein that stimulates the body's adaptive immune system to provide an adaptive immune response. Thus, an antigenic / immunogenic peptide or protein comprises at least one epitope (as defined herein) or antigen (as defined herein) of the protein from which it is derived (e.g., the spike protein (S) of a coronavirus, preferably from SARS-CoV-2 (2019 novel coronavirus).

[0155] As will be appreciated and understood by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of coronavirus antigens of interest. For example, any protein fragment of the novel coronavirus or its mutants, provided that the fragment is immunogenic and confers a protective immune response against the coronavirus; in addition to variants that are identical to the reference protein but truncated, in some embodiments, the antigen comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, and the length of the antigen / antigenic polypeptide can range from about 4, 6, or 8 amino acids to the full-length protein.

[0156] Epitope

[0157] Epitope: As used herein, the term "epitope" (also known in the art as "antigenic determinant") will be recognized and understood by those of ordinary skill in the art to refer to both T cell epitopes and B cell epitopes. A T cell antigen epitope refers to an antigenic epitope recognized by the T cell receptor (TCR). The epitope component is a peptide that has been degraded from a protein and is often found within the antigen molecule. It must be processed by antigen presenting cells (APCs) and then bound to an MHC molecule to form a complex for TCR recognition. It can generally include fragments preferably having a length of about 6 to about 20 or more amino acids, for example. Fragments processed and presented by MHC class I molecules are preferably about 8 to about 10 amino acids in length, such as 8, 9, or 10 (or 11 or 12 amino acids), or fragments processed and presented by MHC class II molecules are preferably about 13 to about 20 or more amino acids in length. These fragments are typically recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule; i.e., these fragments are not typically recognized in their native form. B cell epitopes are generally fragments located on the outer surface of a (native) protein or peptide antigen, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which can be recognized by antibodies, i.e. recognized in their native form. Such epitopes of proteins or peptides can also be selected from any variants of such proteins or peptides mentioned herein. In this context, epitopes can be conformational or discontinuous epitopes, consisting of fragments of proteins or peptides as defined herein, which are discontinuous in the amino acid sequence of the proteins or peptides as defined herein, but are aggregated together in a three-dimensional structure or are continuous or linear epitopes consisting of a single polypeptide chain.

[0158] Nucleic Acids

[0159] The term "nucleic acid" or "nucleic acid molecule" will be recognized and understood by those of ordinary skill in the art. As used herein, the term "nucleic acid" or "nucleic acid molecule" preferably refers to a DNA (molecule) or RNA (molecule). It is preferably used synonymously with the term polynucleotide. Preferably, a nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other via phosphodiester bonds of a sugar / phosphate backbone. The term "nucleic acid molecule" also includes modified nucleic acid molecules, such as base-modified, sugar-modified, or backbone-modified DNA or RNA molecules as defined herein.

[0160] Specifically, the compositions of the present application comprise (at least one) RNA having an open reading frame (ORF) encoding a coronavirus antigen (e.g., a variant trimeric spike protein, e.g., a stabilized pre-fusion spike protein). In some embodiments, the RNA is a messenger RNA (mRNA).

[0161] In some embodiments, the nucleic acid comprises at least one heterologous untranslated region (UTR). The term "untranslated region" or "UTR" or "UTR element" will be recognized and understood by those of ordinary skill in the art to mean a portion of a nucleic acid molecule, typically located 5' or 3' to a coding sequence. The 5' end is referred to as a 5'UTR, and the 3' end is referred to as a 3'UTR. Generally speaking, UTRs are not translated into proteins; UTRs can be part of a nucleic acid, such as DNA or RNA. UTRs can comprise elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements can be ribosome binding sites, miRNA binding sites, etc.; RNA (e.g., mRNA) can further comprise a 5'UTR, a 3'UTR, a 3'-poly(A) and / or a 5' cap analog.

[0162] In some embodiments, the 5'UTR is a heterologous UTR, i.e., a UTR found in nature that is associated with a different ORF; in another embodiment, the 5'UTR is a synthetic UTR; the 5'UTR is a region of the mRNA that is located upstream (5') of the start codon (the first codon of the mRNA transcript translated by the ribosome). The 5'UTR does not encode a protein. The natural 5'UTR has characteristics that play a role in translation initiation, such as the Kozak sequence, which has a consensus CCR(A / G)CCAUGG; exemplary 5'UTRs also include African clawed frog or human α-globin or β-globin, human cytochrome b-245a polypeptide, hydroxysteroid (17b) dehydrogenase, tobacco etch virus, alpha-1-globin 5'UTR, and the like.

[0163] In some embodiments, the 3'UTR can be heterologous or synthetic; for example, globin UTRs, including African clawed frog β-globin UTRs and human β-globin UTRs; other 3'UTRs can also be CYBA (cytochrome b-245alpha chain), rabbit β-globin, hepatitis B virus (HBV), α-globin 3'UTR, and VEEV (Venezuelan equine encephalitis virus) virus 3'UTR sequences. In some embodiments, rps9 (Ribosomal Protein S9) 3'UTR, FIG4 (FIG4 Phosphoinositide 5-Phosphatase), gp130, DH143, and human albumin hHBB (human hemoglobin subunit beta), HBA1 (human Hemoglobin Subunit Alpha 1) 3'UTRs can also be used.

[0164] In some embodiments, the 3'-poly (A) tail is also called a poly (A) tail; the poly (A) tail is located downstream of the 3' UTR, for example, the mRNA region immediately downstream (i.e., 3'), which contains multiple consecutive adenosine monophosphates. The poly (A) tail may contain 10 to 300 adenosine monophosphates, and may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some preferred embodiments, the poly (A) tail contains 50 to 250 adenosine monophosphates, more preferably 50-100 adenosine monophosphates; most preferably 100 adenosine monophosphates; in relevant biological environments (e.g., in cells, in vivo), the function of the 3'-poly (A) tail is to protect the mRNA from enzymatic degradation, for example in the cytoplasm, and to facilitate transcription termination and / or export of the mRNA from the nucleus and translation.

[0165] In some embodiments, the RNA (e.g., mRNA) further comprises a 5' guanosine cap; the 5' guanosine cap is a eukaryotic mRNA transcript, and the 5' cap is composed of an inverted 7-methylguanosine, connected to the rest of the eukaryotic mRNA via a 5'-5' triphosphate bridge, namely cap 0 (cap 0), which primarily serves as a quality control for correct mRNA processing and helps stabilize the eukaryotic mRNA; based on cap 0, the first nucleotide is methylated with 2'-OH, referred to as cap 1 (cap 1); in addition to cap 0 and cap 1, the second nucleotide can also be further methylated, referred to as cap 2; generally speaking, the 5'-cap can be synthesized by different synthetic routes of 5'-capped mRNA based on enzymatic, chemical, or chemoenzymatic methods;

[0166] In some embodiments, during in vitro transcription, a cap analog is directly added to the in vitro transcription (IVT) system, and the 5' cap analog includes but is not limited to: m 7 Gppp(2'OMeA)pG、m 7 GpppApA、m 7 GpppApC、m 7 GpppApG、m 7 GpppApU、m 7 GpppCpA、m 7 GpppCpC、m 7 GpppCpG、m 7 GpppCpU、m 7 GpppGpA、m 7 GpppGpC、m 7 GpppGpG、m 7 GpppGpU、m 7 GpppUpA、m 7 GpppUpC、m 7 GpppUpG、m 7 GpppUpU、m 7 Gpppm 6 ApG、m 7 G 3’Ome pppApA、m 7 G 3’Ome pppApC、m 7 G 3’Ome pppApU、m 7 G 3’Ome pppApG、m 7 G 3’Ome pppCpA、m 7 G 3’Ome pppCpC、m7 G 3’Ome pppCpG、m 7 G 3’Ome pppCpU、m 7 G 3’Ome pppUpA、m 7 G 3’Ome pppUpC、m 7 G 3’Ome pppUpG、m 7 G 3’Ome pppUpU、m 7 G 3’Ome pppA 2’Ome pG、m 7 G 3’Ome pppA 2’Ome pC、m 7 G 3’Ome pppA 2’Ome pU、m 7 G 3’Ome pppA 2’Ome pA、m 7 G 3’Ome pppC 2’Ome pA、m 7 G 3’Ome pppC 2’Ome pU、m 7 G 3’Ome pppC 2’Ome pG、m 7 G 3’Ome pppC 2’Ome pC、m 7 G 3’Ome pppG 2’Ome pA、m 7 G 3’Ome pppG 2’Ome pU、m 7 G 3’Ome pppG 2’Ome pG、m 7 G 3’Ome pppG 2’Ome pC、m 7 G 3’Ome pppU 2’Ome pA、m 7 G 3’Ome pppU 2’Ome pU、m 7 G 3’Ome pppU 2’Ome pG、m 7 G 3’Ome pppU 2’Ome etc.

[0167] In some embodiments, the capped analogs may also be other structures, such as tetramers, pentamers, hexamers, heptamers, octamers, nonamers, or decamers, etc. The specific sequence thereof may be determined according to the conditions of the template.

[0168] It should also be understood that the novel coronavirus vaccine of the present application may include any 5' untranslated region (UTR) and / or any 3' untranslated region (UTR). Nucleic acids comprise polymers of nucleotides (nucleotide monomers). Therefore, nucleic acids are also referred to as polynucleotides. Nucleic acids can be or can include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNAs), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras and / or combinations thereof.

[0169] Messenger RNA (mRNA) is any RNA that encodes (at least one) protein (a naturally occurring, non-naturally occurring, or modified amino acid polymer) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein. The skilled artisan will understand that, unless otherwise indicated, the nucleic acid sequences listed in this application may reference "T" in the representative DNA sequence, but when the sequence represents RNA (e.g., mRNA), the "T" will be replaced with a "U." Therefore, any DNA disclosed and identified herein by a particular sequence identification number also discloses the corresponding RNA (e.g., mRNA) sequence complementary to the DNA, wherein each "T" of the DNA sequence is replaced by a "U."

[0170] open reading frame

[0171] An open reading frame (ORF) is a continuous stretch of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). Generally speaking, an ORF typically encodes a protein. It will be understood that the sequences disclosed herein may also contain additional elements, such as 5' and 3' UTRs, but unlike ORFs, these elements are not necessarily present in the RNA polynucleotides of the present application.

[0172] In some embodiments, the composition comprises RNA (e.g., mRNA) comprising the nucleotide sequence of any one of SEQ ID NOs. 1-19 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity.

[0173] In some embodiments, the open reading frame is preferably at least partially codon optimized. Codon optimization is based on such discovery: translation efficiency can be determined by the different frequencies of transfer RNA (tRNA) occurring in the cell. Therefore, if there is so-called "rare codon" of increasing degree in the coding region of the nucleic acid of the application defined herein, the translation of the corresponding modified nucleotide sequence is less efficient than when there is a codon encoding relatively "common" tRNA. Those skilled in the art can carry out codon optimization for sequence to be translated with the characteristics of its in vitro expression system.

[0174] Chemically modified or unmodified nucleotides

[0175] In some embodiments, RNA (e.g., mRNA) is not chemically modified, but rather comprises standard ribonucleotides consisting of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides disclosed herein comprise standard nucleoside residues, such as those present in transcribed RNA (e.g., A, G, C, or U). In some embodiments, the nucleotides and nucleosides disclosed herein include standard deoxyribonucleosides, such as those present in DNA (e.g., dA, dG, dC, or dT);

[0176] In some embodiments, the compositions of the present application include RNA with an open reading frame encoding a coronavirus antigen, wherein the nucleic acid includes standard (unmodified) or modified nucleotides and / or nucleosides known in the art. In some embodiments, the nucleotides and nucleosides of the present application include modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include modifications of the sugar, backbone or core base moieties of nucleotides and / or nucleosides known in the art.

[0177] In some embodiments, the modified nucleic acid base in the nucleic acid (e.g., RNA nucleic acid, e.g., mRNA nucleic acid) includes 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methoxy-uridine, 5-methyl-cytidine and / or pseudouridine, pseudouridine.

[0178] In vitro transcription system (IVT)

[0179] In vitro transcription is the process of using DNA as a template in an in vitro cell-free system containing components such as RNA polymerase and NTP to mimic the in vivo transcription process to generate mRNA. Generally speaking, the capped RNA synthesized in the in vitro transcription reaction can be used for subsequent experiments such as microinjection, in vitro translation, and transfection. The in vitro transcription system usually includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase. NTPs can be synthesized by oneself or selected from a supplier. NTPs can be natural or non-natural NTPs. Optional polymerases include, but are not limited to, phage RNA polymerases, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or polymerase mutants thereof, such as, but not limited to, polymerases capable of incorporating modified nucleic acids and / or modified nucleotides, including chemically modified nucleic acids and / or nucleotides. Some embodiments exclude the use of DNA enzymes. In some embodiments, the RNA contains a 5' guanosine cap.

[0180] In addition to synthesis by in vitro transcription systems, chemical synthesis methods can also be used, including solid-phase chemical synthesis and liquid-phase chemical synthesis; with respect to solid-phase chemical synthesis, the nucleic acids disclosed in this application can be prepared in whole or in part using solid-phase technology; solid-phase chemical synthesis of nucleic acids is an automated method in which molecules are fixed on a solid support and synthesized stepwise in a reactant solution. Solid-phase synthesis can be used for site-specific introduction of chemical modifications in nucleic acid sequences; with respect to liquid-phase chemical synthesis, the nucleic acids of this application can be synthesized in liquid phase by sequentially adding monomer constructs. In addition, the above-mentioned synthesis methods can also be used in combination, because the synthesis methods discussed above each have their own advantages and limitations, and attempts can be made to combine these methods to overcome the above-mentioned limitations. Combinations of these methods are within the scope of this application.

[0181] Antigenic variants

[0182] In some embodiments, the compositions of the present application comprise RNA encoding coronavirus antigen variants (e.g., variant trimeric spike proteins, such as stabilized pre-fusion spike proteins). Antigen variants or other polypeptide variants refer to molecules whose amino acid sequences are different from wild-type, natural or reference sequences. Compared to natural or reference sequences, antigen / polypeptide variants may have substitutions, deletions and / or insertions at certain positions within the amino acid sequence. Typically, the variant has at least 50% identity with the wild-type, natural or reference sequence. In some embodiments, the variant has at least 80% or at least 90% identity with the wild-type, natural or reference sequence.

[0183] Variant antigens / polypeptides encoded by the nucleic acid of the present application can include amino acid changes that confer any of a variety of desired properties, for example, enhancing their immunogenicity, enhancing their expression and / or improving their stability or PK / PD properties. Conventional mutagenesis techniques can generally be used to prepare variant antigens / polypeptides, and analysis is performed as appropriate to determine whether they have the desired properties. Determining the determination of expression levels and immunogenicity is well known in the art, and exemplary such determinations are set forth in the Examples section. Similarly, techniques generally recognized in the art can be used to measure the PK / PD properties of protein variants, for example, by determining the expression of the antigen in the inoculated subject over time and / or by observing the persistence of the induced immune response. The stability of the protein encoded by the variant nucleic acid can be measured by measuring the thermal stability or stability during urea denaturation, or can be measured using computer prediction. Methods for such experiments and computer determinations are known in the art.

[0184] The term "identity" refers to the relationship between the sequences of two or more polypeptides (e.g., antigens) or polynucleotides (nucleic acids) determined by comparing sequences. Identity also refers to the degree of sequence relatedness between or among sequences determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences, where gap alignments (if any) are solved by a specific mathematical model or computer program (e.g., an "algorithm"). The identities of the related antigens or nucleic acids can be easily calculated by known methods. "Percent (%) identity" for polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid sequence or the nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percentage identity. Methods and computer programs for comparison are well known in the art. It is understood that identity is determined by the calculation of percent identity, but its value may vary due to the gaps and penalties introduced in the calculation. Typically, variants of a particular polynucleotide or polypeptide (e.g., an antigen) have 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a particular reference polynucleotide or polypeptide as determined by the sequence alignment programs and parameters described herein and known to those of skill in the art.

[0185] Lipid nanoparticles (LNPs)

[0186] The RNA of the present application (for example, mRNA) is formulated in lipid nanoparticle (LNP).Lipid nanoparticle generally comprises ionizable cationic lipid, helper lipid, cholesterol and PEG lipid component and nucleic acid of interest.Lipid nanoparticle of the present application can use component generally known in the art, and composition and method generate.

[0187] N:P ratio

[0188] The term "N:P ratio" used in this article refers to the molar ratio of the protonated nitrogen element of the ionizable cationic lipid to the phosphate group of the mRNA. The N:P ratio describes the ratio between the cationic charge of the amino group (N+) in the ionizable cationic lipid and the anionic charge of the phosphate group (PO4-) in the nucleic acid backbone, and is the basis for the electrostatic interaction between the ionizable cationic lipid and the nucleic acid. The N:P ratio is a key formulation factor for LNPs, affecting the physicochemical properties of LNPs and the in vivo release of drugs. Multivalent vaccines

[0189] The compositions provided herein may include RNA or multiple RNAs encoding two or more antigens of the same or different types. In some embodiments, the compositions include RNA or multiple RNAs encoding two or more coronavirus antigens. In some embodiments, the RNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more coronavirus antigens.

[0190] Two or more different RNAs (e.g., mRNA) encoding antigens can be formulated in the same lipid nanoparticle. In other embodiments, two or more different RNAs encoding antigens can be formulated in separate lipid nanoparticles (each RNA is formulated in a single lipid nanoparticle). The lipid nanoparticles can then be combined and administered as a single vaccine composition (e.g., comprising a variety of RNAs encoding multiple antigens), or can be administered separately.

[0191] Combined vaccines

[0192] Compositions provided herein can include RNA or multiple RNAs encoding two or more antigens of the same or different virus strains. Combination vaccines are also provided herein, which include RNA encoding one or more coronaviruses and one or more antigens of different organisms. Therefore, the vaccine of the present application can be a combined vaccine targeting one or more antigens of the same strain / species, or one or more antigens of different strains / species, such as inducing an antigen to which the immunity of the organism found in the same geographic area where the risk of coronavirus infection is high or an individual may be exposed to the coronavirus when being exposed to the coronavirus.

[0193] Sequential inoculation

[0194] Sequential vaccination refers to the interval vaccination of vaccines with different technical routes, including basic immunization sequence and booster immunization sequence; if the first shot is an inactivated vaccine and the second shot is an adenovirus vaccine or mRNA vaccine or any other non-inactivated vaccine, this vaccination method is called basic immunization sequence; if two doses of inactivated vaccine have been completed and a booster shot is needed subsequently, any other non-inactivated vaccine is used instead, this vaccination method is called booster immunization sequence.

[0195] pharmaceutical preparations

[0196] Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for preventing or treating, for example, human and other mammalian coronaviruses. The compositions provided herein can be used as therapeutic or prophylactic agents. They can be used in medicines for preventing and / or treating coronavirus infections.

[0197] The term "pharmaceutical composition" refers to the combination of an active agent and an inert or active carrier such that the composition is particularly suitable for in vivo or in vitro diagnostic or therapeutic use. A "pharmaceutically acceptable carrier" does not cause undesirable physiological effects upon administration to or after administration to a subject. The carrier in a pharmaceutical composition must be "acceptable" in the sense that it is compatible with the active ingredient and capable of stabilizing it. One or more solubilizing agents may be used as pharmaceutical carriers for delivering the active agent. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible carriers, adjuvants, additives, and diluents to obtain a composition that can be used as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Other suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for them, are described in Remington's Pharmaceutical Sciences.

[0198] As used herein, the term "about" refers to the usual error range for each value that is readily known to those skilled in the art. Reference to an "about" value or parameter in this article includes embodiments for the value or parameter itself. As used herein, when the term "about" precedes a numerical value, it generally means within a range of 10% above or below that numerical value. For example, "about 100" encompasses 90 and 110. In some embodiments, when "about" is followed by an integer not greater than 10, the decimals are included, and the integer can be obtained by rounding the decimal values ​​of these decimals. For example, about 9 includes the endpoint values ​​of the range 8.5 to 9.5 and all values ​​therebetween.

[0199] The relevant sequences involved in this application are as follows:

[0200] It should be understood that the following sequences are only exemplary sequences of embodiments of the present application, and are not any limitation to the embodiments of the present application. The nucleic acid sequence in the following sequence may represent a DNA sequence or an RNA sequence. When it represents an RNA sequence, unless otherwise specified, the "T" therein represents uridine. It should be understood that in this application, unless otherwise specified, the "uridine" of this application covers natural uridine and its derivatives, including but not limited to: 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methylcytidine, 5-methoxycytidine, 1-methyl pseudouridine (N1-Methyl-Pseudo-UTP), pseudouridine, 1-ethyl-pseudouridine, and 5-methoxy-uridine. In other embodiments, all or part of the nucleic acid of the present application can be replaced with a modified base, such as 1-methylpseudouridine or pseudouridine.

[0201] The embodiments of the present application will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present application and should not be considered as limiting the scope of the present application. In the examples, if no specific conditions are specified, the conditions according to conventional conditions or manufacturer recommendations are used. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0202] The sequences used in the following examples correspond to the specific sequences listed above with sequence numbers or sequence names.

[0203] Example 1: Codon optimization and in vitro transcription (IVT) of S protein antigen

[0204] 1.1 Codon optimization experiments

[0205] According to the natural coding region sequence of the novel coronavirus Omicron S protein, mutations and optimizations were carried out on its basis, and the SF mRNA sequence was designed and synthesized (SEQ ID NO.1-SEQ ID NO.19, where SF mRNA refers to the full-length mRNA of the S protein). In addition to the coding region (ORF), the SF mRNA sequence features also include 5'UTR, 3'UTR and polyA. Compared with SF-0 (the original Wuhan-hu-1S protein coding sequence, NC_045512.2), the GC content of the mRNA of the S protein that has not been codon-optimized has increased. The results of the expression experiment showed that the mRNA expression levels of all codon-optimized Omicron strains were better than those of the control (without codon optimization). The following are some of the results as examples:

[0206] Table 1: mRNA relative expression data

[0207] Note: SF-0 is the mRNA sequence encoding the S protein of the Wuhan-hu-1 strain without codon optimization.

[0208] 1.2 In vitro transcription (IVT)

[0209] 1.2.1 According to the instructions of the IVT kit (E131, Novoprotein), the IVT reaction system was prepared by mixing 10× Transcription Buffer, ATP, GTP, CTP, 1-N-Me-Pseudo UTP (Cat. No. WA0992, Zhaowei Technology), 5' cap analog m7G(5')ppp(5')(2'OMeA)pG (Cat. No. GAGNH23C2L1B, Zhaowei Technology), water for injection, linearized plasmid template of T7 promoter (customized by GenScript Biotech Co., Ltd., the template contains the DNA sequence corresponding to SEQ ID NOs. 1-19), and Enzyme Mix;

[0210] 1.2.2 The mixed reaction system was incubated at 37°C for 65 min;

[0211] 1.2.3 Add the appropriate proportion of DNase I to terminate the reaction.

[0212] After in vitro synthesis, S protein mRNA was separated and purified by hydrophobic chromatography and ultrafiltration concentration to obtain high-purity mRNA (>95%). The specific results are shown in Figure 1. The experimental results show that the above steps have obtained high-purity mRNA, which can be used for subsequent experiments. Note: Internal standard (25nt, Agilent, RNA 6000 Nano Kit (reorder-no 5067-1511)

[0213] Example 2: Preparation and confirmation of protein antigen mRNA-LNP formulation

[0214] 1. Accurately weigh a certain amount of lipids with a molar mass ratio of 50% SM-102, 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG2000 and dissolve them in an appropriate amount of anhydrous ethanol to prepare a lipid working solution (final concentration of lipid working solution 20 mg / mL).

[0215] 2. Prepare 130 mM sodium chloride in citric acid buffer (10 mM, pH 4.0), Tris-NaOAc buffer (20 mM, 10.7 mM, pH 7.5), and 60% sucrose in Tris-NaOAc buffer (20 mM, 10.7 mM, pH 7.5).

[0216] 3. Take an appropriate amount of the mRNA stock solution synthesized and purified in Example 1 and dilute it with the sodium chloride-citrate buffer solution prepared above to adjust the final concentration of the mRNA working solution to 0.18 mg / mL.

[0217] 4. Using a microfluidic instrument and supporting chip (shm, Myana (Shanghai) Instrument Technology Co., Ltd.), the lipid working solution and the mRNA working solution were mixed in a volume ratio of 1:3 to prepare the mRNA-loaded LNP solution.

[0218] 5. Dilute the prepared LNP solution by adding 9 times the volume of Tris-NaOAc buffer solution, and concentrate and purify it by tangential flow ultrafiltration (TFF) to remove the ethanol solution in the system.

[0219] 6. Use UV spectrometry to detect the mRNA content in the LNP solution and add an appropriate amount of Tris-NaOAc buffer solution (20mM, 10.7mM, pH 7.5) containing 60% sucrose to adjust the final mRNA concentration in the finished LNP solution to 100μg / mL. At the same time, the sucrose content in the external aqueous phase system is 8.7%.

[0220] 7. S protein mRNA and lipid working solution are mixed by microfluidics to form LNP complex.

[0221] The experimental results showed that the complex morphology of the S protein mRNA and lipid nanoparticles (LNPs) was fully fused, forming thermodynamically stable finished lipid nanoparticles with a particle size of approximately 100 nm, good uniformity, and consistent morphology. After 6 months of refrigeration (5°C ± 3°C), 14 days of storage at room temperature (25°C ± 2°C), and 12 months of storage at frozen conditions (-20°C ± 5°C), the particles showed no significant changes in their physical and chemical properties and biological activity, demonstrating superior thermal stability compared to similar products.

[0222] Example 3: Immunoblotting and ELISA experiments on S protein expression in host cells

[0223] The S protein (including: BQ.1, XBB, BA.5, BF.7, XBB.1.5 and XBB.1.16, etc.) mRNA-LNP of different Omicron strains prepared in Example 2 was transfected into COS-7 cells (African green monkey kidney fibroblasts, purchased from ATCC). After 24 hours of transfection, the cells were collected and subjected to immunoblotting and ELISA detection. Cells not transfected with S protein mRNA were used as negative controls to verify that the cell lysate containing S protein and S1 protein (the product of enzymatic hydrolysis of the Flin site) was a positive control. β-Actin was used as an internal reference. The results show (see Figures 2-7, Figure A is an immunoblot, and Figure B is an ELISA test): the full-length S protein and S1 subunit protein of the mRNA expression products designed for different strains can be detected, and their purity meets the requirements of further experiments.

[0224] Example 4: Antibody Neutralization Pseudovirus Experiment

[0225] 4.1 The steps for preparing the pseudovirus of the new coronavirus are as follows:

[0226] 4.1.1 HEK293T cells were transfected simultaneously with expression plasmids for the SARS-CoV-2 S protein and its mutants and an HIV-1 backbone plasmid (GeneScript Biotech Co., Ltd.) using polyethyleneimine (PEI). The HIV-1 backbone plasmid simultaneously expresses the viral packaging protein and the luciferase reporter gene.

[0227] 4.1.2 After 48 or 72 hours of transfection, the supernatant containing the pseudovirus was collected and filtered through a 0.45 μm filter to remove cell debris to obtain pseudoviruses (SARS-CoV-2-Fluc BQ.1, i.e., a pseudovirus containing the S protein of BQ.1 on the envelope; SARS-CoV-2-Fluc XBB, i.e., a pseudovirus containing the S protein of XBB on the envelope; SARS-CoV-2-Fluc BF.7, i.e., a pseudovirus containing the S protein of BF.7 on the envelope; SARS-CoV-2-Fluc BA.4 / BA.5, i.e., a pseudovirus containing the S proteins of BA.4 and BA.5 on the envelope).

[0228] 4.1.3 Aliquot the pseudovirus supernatant obtained by filtration and store at -196°C.

[0229] 4.1.4 The pseudovirus titer was detected using p24 ELISA kit (632200, Takara) and confirmed to meet the requirements for further experiments.

[0230] 4.2 In vitro binding assay and in vivo pseudovirus neutralizing antibody assay

[0231] 4.2.1 In vitro binding assay

[0232] The serum of mice (BALB / c, half male and half female, 6-8 weeks old) immunized with the S protein mRNA-LNP preparation (prepared in Example 2) was incubated with S protein (including S protein of Omicron BA.5, Omicron BF.7, Omicron BQ.1, and Omicron XBB strains, all commercially available), and the binding ability of mouse serum antibodies to S protein was detected by ELISA.

[0233] 4.2.2 Pseudovirus neutralizing antibody experiment

[0234] The luciferase-labeled SASR-CoV-2 pseudovirus (including pseudoviruses of Omicron BA.5, Omicron BF.7, Omicron BQ.1, and Omicron XBB strains) was co-incubated with the serum of the above-mentioned S protein mRNA-immunized mice. The culture medium after the above co-incubation was then used to infect HEK293T cells constitutively expressing ACE2 protein. The fluorescence value of the intracellular pseudovirus was detected 48 hours after transfection, and the pseudovirus neutralization titer was calculated. The control group (Control) was an unimmunized mouse serum group. Among them, ID 50 NT is the median infectious dose; 50 is the half-neutralization titer.

[0235] Based on the above experimental results: Figures 8-11 respectively show the relevant experimental results for different strains (Omicron BA.5, Omicron BF.7, Omicron BQ.1, Omicron XBB) (for each strain, Figure A shows the in vitro binding experiment results, and Figure B shows the pseudovirus neutralizing antibody experiment). The results show that the mRNA vaccine prepared for the above strains can effectively produce antibodies with high binding ability to the S protein, and the relevant antibodies can effectively neutralize the pseudovirus.

[0236] Example 5: Bivalent vaccine pseudovirus neutralizing antibody experiment

[0237] The luciferase-labeled SASR-CoV-2 pseudovirus was co-incubated with serum from immunized mice (BALB / c mice, half male and half female, 6-8 weeks old) (i.e., serum from mice immunized with Omicron XBB mRNA-LNP (SEQ ID NO.6) and BQ.1 mRNA-LNP (SEQ ID NO.10)). The culture medium after the co-incubation was then used to infect HEK293T cells constitutively expressing ACE2 protein. 48 hours after transfection, the fluorescence value of the intracellular pseudovirus was detected, and the pseudovirus neutralization titer was calculated. The specific immunization scheme and grouping are as follows:

[0238] Mice were immunized on days 0 and 14, and serum was collected for testing on day 21. Mice were divided into the following experimental groups, with 5 mice in each experimental group.

[0239] BQ.1 group: mice were immunized with 3 μg of mRNA-LNP preparation expressing the S protein of the BQ.1 strain;

[0240] XBB group: mice were immunized with 3 μg of mRNA-LNP preparation expressing the S protein of the XBB strain;

[0241] Combined immunization group (separate): mice were injected simultaneously (into the left and right thigh muscles) with mRNA-LNP preparations targeting the two strains mentioned above, with an injection dose of 1.5 μg for each strain;

[0242] Bivalent mixed injection group: LNPs containing 1.5 μg BQ.1 and 1.5 μg XBB strains were mixed at a ratio of 1:1 and injected intramuscularly to immunize mice

[0243] The experimental results show (Figure 12): Compared with monovalent vaccines, the immunization method using bivalent vaccines can significantly increase the content of neutralizing antibodies against the two strains, regardless of whether it is mixed or injected simultaneously; in addition, compared with simultaneous injection, mixed injection has better effect.

[0244] Example 6: Pharmacology and pharmacodynamics studies of bivalent vaccines

[0245] To further verify the synergistic immune effect of BQ.1 and XBB lineage S protein, the following experiments were conducted.

[0246] 6.1BQ.1+XBB vaccine immunogenicity

[0247] Male BALB / c mice, 6 to 8 weeks old, were immunized intramuscularly with 3 μg / mouse of either BQ.1 vaccine (i.e., BQ.1 mRNA-LNP, where the mRNA sequence is shown in SEQ ID NO: 10) or XBB vaccine (i.e., XBB mRNA-LNP, where the mRNA sequence is shown in SEQ ID NO. 6). A mixed group (mixed vaccine immunization group) received one injection of 1.5 μg of BQ.1 vaccine or 1.5 μg of XBB vaccine. A 1 / 10 dose of a Chinese inactivated vaccine (inactivated novel coronavirus (CZ02 strain), 0.5 ml per human dose, containing 600 SU of inactivated novel coronavirus antigen) was used as a control. Immunizations were performed twice, 14 days apart (denoted as D0 and D14, respectively). Serum was tested for pseudovirus neutralization activity 7 days after the second immunization (denoted as D21). Serum was tested for true and pseudovirus neutralization activity 14 days after the second immunization (i.e., D28).

[0248] Pseudovirus neutralization studies used pseudoviruses derived from Omicron variants (BQ.1.1, XBB, and XBB.1.5), and serum was collected on day 21 (7 days after two injections) for testing. The test serum was incubated with pseudoviruses derived from SARS-CoV-2 Omicron variants (BQ.1.1, XBB, and XBB.1.5), and then infected with hACE2-overexpressing cells. The neutralizing antibody titers of the test serum samples on the ACE2-overexpressing cells were measured. The results showed that at the same 1 / 10 dose, the neutralizing activity of serum from the BQ.1, XBB, and mixed vaccine immunization groups against Omicron variants (BQ.1.1, XBB, and XBB.1.5) was significantly superior to that of the inactivated vaccine marketed in China. BQ.1 and XBB vaccines provided superior protection against the target strains, and mixed immunization provided superior cross-protection against the Omicron strain compared to the same dose of a single S protein mutant mRNA vaccine (see Figure 13 for details).

[0249] Virus neutralization studies (CPE) were conducted using real viruses from Omicron variants (BA.5, BF.7, BQ.1, and XBB), and serum was collected on day 28 (14 days after two doses). The results showed that the BQ.1 and XBB vaccines, as well as the mixed immunization group, cross-protected against Omicron variants (BA.5, BF.7, BQ.1, and XBB). At the same 1 / 10 dose, the neutralizing activity of sera from the BQ.1, XBB, and mixed immunization groups against Omicron variants (BA.5, BF.7, BQ.1, and XBB) was significantly superior to that of the aforementioned inactivated vaccine marketed in China. The BQ.1 and XBB vaccines provided superior protection against the target strains, and the cross-protection of mixed immunization against Omicron strains was superior to that of the same dose of a single iterative vaccine. See Figure 14 for details.

[0250] 6.2 Immunogenicity of BQ.1+XBB.1.5 Vaccine

[0251] This section confirms that the immunogenicity induced by mRNA mixture 1 (a mixed preparation of BQ.1 mRNA-LNP and XBB.1.5 mRNA-LNP, wherein the sequence of the mRNA in BQ.1 mRNA-LNP is shown in SEQ ID NO: 10, and the sequence of the mRNA in XBB.1.5 mRNA-LNP is shown in SEQ ID NO: 15) against the currently prevalent Omicron strains (BA.5, BQ.1, XBB.1.5 and XBB.1.16, XBB.1.9 and EG.5) is significantly better than that of SYS6006 (the pharmaceutical change relative to mRNA mixture 1 is limited to the replacement of the antigen sequence with BA.5 mRNA, that is, SYS6006 is BA.5 mRNA-LNP).

[0252] Six-week-old BALB / c mice were selected and divided into 10 groups, half male and half female. Each group was intramuscularly injected with 3μg / mouse SYS6006, 3μg / mouse mRNA mixture 1, 3μg / mouse XBB.1.5 mRNA-LNP preparation or 3μg / mouse BQ.1 mRNA-LNP preparation. The difference between the groups was limited to the replacement of the antigen sequence. Immunization was performed twice with an interval of 21 days. Serum was collected 14 days after the second immunization (i.e., D35) to detect the neutralizing activity of true virus and pseudovirus. Serum was collected before the second immunization, 14 days after the second immunization, and 21 days after the second immunization to detect specific IgG binding antibodies and compare the immunogenicity of the bivalent new coronavirus mRNA vaccine and the monovalent vaccine.

[0253] Specific IgG antibody titers were measured in mouse serum using an ELISA method to measure S protein-specific IgG antibody titers. Binding antibody results showed that within the blood collection period, binding antibody titers in serum increased to 1:1e5 or above after a single immunization with SYS6006 or mRNA mixture 1, and increased by at least one order of magnitude after a double immunization. There was no difference in serum S protein-specific IgG antibody levels after immunization with equal doses of SYS6006 and mRNA mixture 1. Furthermore, with increasing immunization doses, binding antibody levels in serum from SYS6006 and mRNA mixture 1 increased modestly, as shown in Figure 15. Overall, these results indicate that mRNA mixture 1 and SYS6006 do not differ in their ability to elevate serum specific binding antibody levels.

[0254] The CPE method was used to detect the neutralization of multiple real virus strains including BA.5, BQ.1, XBB.1.5 and XBB.1.16 by immune serum. IC50 was calculated. The results showed that the mRNA mixture 1 immune serum had a strong cross-protective effect on BA.5, BQ.1, XBB.1.5, XBB.1.16, XBB.1.19 and EG.5 real viruses. With the increase of the immune dose, the mRNA mixture 1 had a strong cross-protective effect on the neutralization of each strain. The antibody level increased to a certain extent. Under the same dose conditions, the neutralizing activity of mRNA mixture 1 immune serum against various variants (BA.5, BQ.1, XBB.1.5, XBB.1.16, XBB.1.19 and EG.5) was significantly better than that of SYS6006, especially the geometric mean titer (GMT) of neutralizing antibodies against the existing epidemic strains XBB.1.5 and EG.5 could reach 100 times. Under the same dose conditions, the neutralizing activity of mRNA mixture 1 against BQ.1 was slightly better than that of BQ.1. The mRNA-LNP preparation was significantly superior to the XBB.1.5 mRNA-LNP preparation (P<0.01); its neutralizing activity against XBB.1.5, XBB.1.16, XBB.1.19, and EG.5 was slightly superior to that of the XBB.1.5 mRNA-LNP preparation, and significantly superior to that of the BQ.1 mRNA-LNP preparation (P<0.05), suggesting that the bivalent SARS-CoV-2 mRNA vaccine (mRNA mixture 1) has superior cross-protection against viruses compared to the monovalent vaccine, and that the XBB.1.5 and BQ.1 mRNAs have a synergistic effect in cross-protection. See Figure 16 for details.

[0255] In the in vitro neutralizing activity study of pseudoviruses, the test serum was incubated with SARS-CoV-2 Omicron mutant strains (BA.4 / 5, BQ.1, XBB.1.5, XBB.1.16 and EG.5) pseudoviruses, and then infected hACE2-overexpressing cells to determine the neutralizing antibody titer of the test serum samples on ACE2 cells. Results showed that serum immunized with mRNA mixture 1 exhibited strong cross-protection against pseudoviruses BA.4 / 5, BQ.1, XBB.1.5, XBB.1.16, and EG.5, with neutralizing antibody levels increasing with increasing immunization doses. Under equal dose conditions, serum immunized with mRNA mixture 1 exhibited significantly superior neutralizing activity against each variant (BA.4 / 5, BQ.1, XBB.1.5, XBB.1.16, and EG.5) compared to SYS6006. In particular, the geometric mean titers (GMTs) against the currently circulating XBB.1.5, XBB.1.16, and EG.5 strains reached 1041-fold, 1127-fold, and 1311-fold, respectively. See Figure 17 for details.

[0256] In summary, the immune serum of mRNA mixture 1 has cross-protection against the existing epidemic strains Omicron (BA.5, BQ.1, XBB.1.5 and XBB.1.16, XBB.1.9 and EG.5), and the cross-protection effect is better than that of SYS6006. The mRNAs of XBB.1.5 and BQ.1 in mRNA mixture 1 have a synergistic effect in the cross-protection effect of the virus.

[0257] This indicates that mRNA mixture 1 and SYS6006 showed no difference in their ability to elevate serum-specific binding antibody levels. Sera immunized with mRNA mixture 1 demonstrated superior cross-protection against the major circulating Omicron strains (BA.5, BQ.1, XBB.1.5, XBB.1.16, and the major circulating strain EG.5) compared to SYS6006.

[0258] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. An mRNA encoding a coronavirus antigen comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 30-37, or the amino acid sequence of the antigen is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 30-37.

2. The mRNA of claim 1, comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-19, or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-19.

3. An mRNA mixture comprising mRNA of at least two S protein mutants of the new coronavirus, wherein the at least two S protein mutants are selected from any two of the following: S proteins of BQ.1, XBB, BA.5, BF.7, XBB.1.5 and XBB.1.

16.

4. The mRNA mixture of claim 3, wherein the mRNAs of the at least two S protein mutants respectively comprise a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-19, or its sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-19.

5. The mRNA mixture as described in claim 3 or 4, wherein the at least two S protein mutants comprise: an S protein of BQ.1 and an S protein of XBB.1.

5.

6. The mRNA mixture of any one of claims 3-5, wherein the sequences of at least two of the S protein mutant mRNAs are identical to SEQ ID NO: 10 and SEQ ID NO: 15, or are at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 10 and SEQ ID NO:

15.

7. The mRNA according to claim 1 or 2, or the mRNA mixture according to any one of claims 3 to 6, wherein the mRNA comprises a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR) and a 3'-poly(A), preferably the 3'-poly(A) comprises 50 to 200 adenosines.

8. The mRNA according to claim 1 or 2, or the mRNA mixture according to any one of claims 3 to 6, wherein the mRNA comprises a 5'UTR, a 3'UTR and a 3'-poly(A), wherein the 3'-poly(A) comprises the nucleotide sequence of SEQ ID NO.

24.

9. The mRNA according to claim 1 or 2, or the mRNA mixture according to any one of claims 3 to 6, wherein the mRNA comprises a 5'UTR, a 3'UTR and a 3'-poly(A), the 5'UTR comprising a nucleotide sequence selected from any one or more of SEQ ID NOs: 21 to 23 and / or the 3'UTR comprising a nucleotide sequence selected from any one or more of SEQ ID NOs: 25 to 28; preferably, the 5'UTR is composed of a sequence as shown in SEQ ID NO: 21 and SEQ ID NO: 22 connected in series from 5' to 3', and the 3'UTR is composed of a sequence as shown in SEQ ID NO: 25 and SEQ ID NO: 26 connected in series from 5' to 3'.

10. The mRNA of claim 1 or 2, or the mRNA mixture of any one of claims 3 to 6, wherein the mRNA comprises a 5'UTR, a 3'UTR and a 3'-poly(A), and the mRNA further comprises a 5' guanosine cap selected from any one of the following: m7Gppp(2'OMeA)pG、m7GpppApA、m7GpppApC、m7GpppApG、m7GpppApU、m7GpppCpA、m7GpppCpC、m7GpppCpGpp、m7GpCpC 、m7GpppGpA、m7GpppGpC、m7GpppGpG、m7GpppGpU、m7GpppUpA、m7GpppUpC、m7GpppUpG、m7GpppUpU、Ap6GppmGpG 3’Ome pppApA、m7G 3’Ome pppApC、m7G 3’Ome pppApU、m7G 3’Ome pppApG、m7G 3’Ome pppCpA、m7G 3’Ome pppCpC、m7G 3’Ome pppCpG、m7G 3’Ome pppCpU、m7G 3’Ome pppUpA、m7G 3’Ome pppUpC、m7G 3’Ome pppUpG、m7G 3’Ome pppUpU、m7G 3’Ome PppA 2’Ome pG、m7G 3’Ome pppA 2’Ome pC、m7G 3’Ome pppA 2’Ome pU、m7G 3’Ome pppA 2’Ome pA、m7G 3’Ome pppC 2’Ome pA、m7G 3’Ome pppC 2’Ome pU、m7G 3’Ome pppC 2’Ome pG、m7G 3’Ome pppC 2’Ome pC、m7G 3’Ome pppG 2’Ome pA、m7G 3’Ome pppG 2’Ome pU、m7G 3’Ome pppG 2’Ome pG、m7G 3’Ome pppG 2’Ome pC、m7G 3’Ome pppU 2’Ome pA、m7G 3’Ome pppU 2’Ome pU、m7G 3’Ome pppU 2’Ome pG、m7G 3’Ome U 2’Ome pC; preferably m7Gppp(2'OMeA)pG.

11. mRNA as claimed in claim 1 or 2, or according to the mRNA mixture according to any one of claims 3-6, wherein the coronavirus antigen encoded by the mRNA comprises at least one of the following mutations relative to the S protein of the wild-type strain (NCBI sequence number: NC_045512.2): Spike_T19I, Spike_L24del, Spike_P25del, Spike_P26del, Spike_A27S, Spike_H69del, Spike_V70del, V83A, Spike_G142D, Spike_H146Q, Spike_W152R, Spike_F157L, Spike_E180V, Spike_Q183E, Spike_I210V, Spike_V213G, Spike_G252V, Spike_G257S, Spike_G339D, Spike_L368I ,Spike_S371F,Spike_S373P,Spike_S375F,Spike_T376A,Spike_D405N,Spike_R408S,Spike_K417N ,Spike_N440K,Spike_K444T,Spike_V445P,Spike_L452R,Spike_N460K,Spike_S477N,Spike_T478K ,Spike_E484A,Spike_F486V,Spike_F490S,Spike_Q498R,Spike_N501Y,Spike_Y505H,Spike_D614G ,Spike_H655Y,Spike_N679K,Spike_P681H,Spike_N764K,Spike_D796Y,Spike_Q954H,Spike_N969K.

12. A composition comprising lipid nanoparticles and messenger RNA (mRNA), wherein the mRNA comprises or is the mRNA described in any one of claims 1-2 and 7-11 or the mRNA mixture described in any one of claims 3-11.

13. The composition of claim 12, wherein the lipid nanoparticles comprise an ionizable cationic lipid, a structural lipid, a helper lipid, and a surfactant.

14. The composition of claim 13, wherein the ionizable cationic lipid is selected from any one or more of the following: SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200 and DlinDMA, preferably the ionizable cationic lipid is SM-102.

15. The composition of any one of claims 13 or 14, wherein the structured lipid is selected from cholesterol, and cholesterol derivatives, preferably cholesterol.

16. The composition according to any one of claims 13 to 15, wherein the helper lipid is selected from any one or more of the following: DSPC, DOPE, DOPC, DOPG and DOPS, preferably the helper lipid is DSPC.

17. The composition according to any one of claims 13 to 16, wherein the surfactant is selected from any one or more of the following: PEG2000-DMG, PEG-DSPE, DTDA-PEG2000, and TPGS, preferably the surfactant is PEG2000-DMG.

18. The composition of any one of claims 12 to 17, wherein the lipid nanoparticles comprise, in terms of molar percentage (mol%), 20-60 mol% ionizable cationic lipids, 25-55 mol% structural lipids, 5-25 mol% auxiliary lipids and 0.5-15 mol% surfactants.

19. The composition of claim 18, wherein the lipid nanoparticles comprise 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG.

20. The composition of any one of claims 13-19, wherein the molar ratio of the protonable nitrogen element to the phosphate groups of the mRNA in the lipid nanoparticles (N:P ratio) is about 2:1 to about 30:1; preferably about 3:1 to about 6:1; and more preferably an N:P ratio of about 6:

1.

21. The mRNA of any one of claims 1-2 and 7-11, the mRNA mixture of any one of claims 3-11, or the composition of any one of claims 12-20, wherein the mRNA comprises a chemically modified base or analog, wherein the chemically modified base or analog is selected from one or more of the following: 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, 1-methyl pseudouridine (N1-Methyl-Pseudo-UTP) and pseudouridine; preferably, the chemically modified base or analog is 1-methyl pseudouridine; further preferably, the uridine in the mRNA sequence is all 1-methyl pseudouridine.

22. The composition of claim 21, comprising at least two mRNAs, wherein the at least two mRNAs are encapsulated in different lipid nanoparticles, or are mixed in a certain ratio and then encapsulated in the same lipid nanoparticle.

23. The composition of any one of claims 12-22, wherein the lipid nanoparticles have an average diameter of from about 50 nm to about 150 nm; preferably have an average diameter of from about 70 nm to about 120 nm; further preferably about 100-120 nm; most preferably about 100 nm.

24. The composition of any one of claims 12-23, further comprising an external phase buffer.

25. The composition of claim 24, wherein the external phase buffer comprises: Tromethamine, sodium acetate, sucrose, pH=7-8; the content of tromethamine is 10-30mmol / L, preferably about 15-25mmol / L, preferably about 15mmol / L, 15.5mmol / L, 16mmol / L, 16.5mmol / L, 17mmol / L, 17.5mmol / L, 18mmol / L, 18.5mmol / L, 19mmol / L, 19.5mmol / L, 20mmol / L, 20.5mmol / L, 21mmol / L, 21.5mmol / L, 22mmol / L, 22.5mmol / L, 23mmol / L, 23.5mmol / L, 24mmol / L, 24.5mmol / L or 25mmol / L, most preferably about 20mmol / L; The content of sodium acetate is 0-20mmol / L, preferably about 5-11mmol / L, preferably about 5mmol / L, 5.5mmol / L, 6mmol / L, 6.5mmol / L, 7mmol / L, 7.5mmol / L, 8mmol / L, 8.5mmol / L, 9mmol / L, 9.5mmol / L, 10mmol / L, 10.5mmol / L, 10.6mmol / L, 10.7mmol / L, 10.8mmol / L, 10.9mmol / L, 11mmol / L, 11.5mmol / L, 12mmol / L, 12.5mmol / L or 13mmol / L, most preferably about 10.7mmol / L; The sucrose content (w / v) is: 5-15%, preferably about 7.5-10%, more preferably about 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 9%, 9.5% or 10%, most preferably about 8.7%.

26. The composition of claim 25, comprising mRNA having a sequence as shown in SEQ ID NO: 10 and SEQ ID NO: 15, lipid nanoparticles, and an external phase buffer; The external phase buffer comprises: about 20 mmol / L tromethamine, about 10.7 mmol / L sodium acetate, about 8.7% (w / v) sucrose, and has a pH of about 7.4; The lipid nanoparticles contain: about 50 mol % SM-102, about 10 mol % DSPC, about 38.5 mol % cholesterol and about 1.5 mol % PEG2000-DMG, with an N:P ratio of about 6.

27. Use of the mRNA of any one of claims 1-2 and 7-11, the mRNA mixture of any one of claims 3-11, or the composition of any one of claims 12-26 in the preparation of a medicament for preventing and / or treating infection by the new coronavirus SARS-CoV-2, preferably, the medicament is a vaccine.

28. The use according to claim 27, wherein the vaccine is a multi-combination vaccine or a multivalent vaccine.

29. The use of claim 28, wherein the vaccine is a SARS-CoV-2 virus mRNA vaccine, and preferably the vaccine is a SARS-CoV-2 virus mutant vaccine, wherein the mutant is preferably selected from any one or more of the following: B.1.1.7 (British mutant, Alpha strain, key mutations: D614G, N501Y, P681H), B.1.351 (South African mutant, Beta strain, key mutations: E484K, N501Y, K417N), B.1.617.1 (first generation Indian mutant, Kappa strain, key mutations: L452R, E484Q, D 614G), B.1.617.2 (second-generation Indian mutant strain, Delta strain, key mutations: L452R, E484Q, D614G), P.1 (Brazilian mutant strain, key mutations: K417T, E484K, N501Y), and Omicron strains BA.5, BA.2.75, BQ.1, BF.7, XBB, XBB.1.5, CH.1.1, XBB.1.16; further preferably, the mutant strains are selected from any one or more of the following: BA.4, BA.5, BQ.1, XBB.1.5, XBB.1.16, XBB.1.9 and EG.

5.

30. A method for preventing a condition caused by SARS-CoV-2 virus infection in a subject, comprising administering to the subject a composition according to any one of claims 12 to 26.

31. A method for inducing a neutralizing antibody response against SARS-CoV-2 in a subject, comprising administering to the subject a composition according to any one of claims 12-26.

32. A combination vaccine comprising a first vaccine and a second vaccine used sequentially, wherein the first vaccine comprises the mRNA described in any one of claims 1-2 and 7-11, the mRNA mixture described in any one of claims 3-11, or the composition described in any one of claims 12-26.

33. The combination vaccine of claim 32, wherein the second vaccine is selected from any one or more of the following: attenuated or inactivated vaccines, adenovirus vaccines, mRNA vaccines, DNA vaccines, and recombinant protein vaccines.

34. The combination vaccine of claim 32 or 33, wherein the second vaccine is selected from any one or more of the following: Moderna (mRNA-1273), CureVac (CVnCoV), Johnson & Johnson (COVID-19 Vaccine Janssen), AstraZeneca (Vaxzevria), Pfizer / BioNTech (Comirnaty), Sputnik (Gam-COVID-Vac), Sinovac (COVID-19 Vaccine (Vero Cell), Novavax (NVX-CoV2373), Shijiazhuang Pharmaceutical Group's new crown mRNA vaccine SYS6006, CanSino's new crown vaccine, Beijing Sinopharm's new crown vaccine, and the Chinese Academy of Sciences-Zhifei's recombinant new coronavirus vaccine.

35. A bivalent vaccine comprising a first mRNA and a second mRNA, characterized in that: 1) the first mRNA comprises a first reading frame encoding a first antigen protein, and the second mRNA comprises a second reading frame encoding a second antigen protein, and the first mRNA molecule and the second mRNA molecule are respectively encapsulated in different lipid nanoparticles; or 2) the first mRNA comprises a first reading frame encoding a first antigen protein, and the second mRNA comprises a second reading frame encoding a second antigen protein, and the first mRNA molecule and the second mRNA molecule are mixed in a certain ratio and then encapsulated in the same lipid nanoparticle; Furthermore, the first mRNA molecule and the second mRNA molecule each independently contain a nucleotide sequence as shown in any one of SEQ ID NOs: 1-19, or each independently consist of a nucleotide sequence as shown in any one of SEQ ID NOs: 1-19, and the lipid nanoparticles contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG.

36. Use of the combination vaccine according to any one of claims 32 to 34 and the bivalent vaccine according to claim 35 in the preparation of a medicament for preventing and / or treating infection by the new coronavirus SARS-CoV-2.

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