Novel Coronavirus Vaccines, Their Preparation Methods, and Applications

Abstract

This disclosure provides a novel coronavirus vaccine, its preparation method, and its application, relating to the technical field of vaccines. The novel coronavirus vaccine contains a nucleic acid molecule encoding the BA.5 S protein of the novel coronavirus Omega occulta variant.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese patent application filed on September 30, 2022 (application number: 2022112076839, invention title: novel coronavirus vaccine and its preparation method and application), the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to the field of vaccine technology, and in particular to a novel coronavirus vaccine, its preparation method, and its application. Background Technology

[0004] The novel coronavirus is highly prone to mutation. Since its discovery, various strains of the novel coronavirus have emerged, including the original strain, the Alpha strain, the Beta strain, the Gamma variant, the Kappa strain, the Delta strain, and the Omicron strain.

[0005] Currently marketed and most COVID-19 vaccines in clinical trials are designed with antigens targeting the original strain of the SARS-CoV-2 virus. Compared to the original SARS-CoV-2 strain sequence, the Omicron variant has at least 60 new mutations, including more than 35 mutations in the spike protein (S protein), with 15 mutations in the most critical receptor-binding domain of the S protein. In contrast, the Delta variant has only 2 mutations in this region. Based on the differences in mutation sites, the Omicron variant can be divided into at least 5 sub-variants: BA.1, BA.2.12.1, BA.2, BA.4, and BA.5.

[0006] Studies have found that the protective efficacy of currently available vaccines against variant strains has decreased to varying degrees, especially against the Omega strain of the novel coronavirus. Therefore, the development of a COVID-19 vaccine with better protective efficacy against variant strains is urgently needed. Summary of the Invention

[0007] According to various embodiments of this disclosure, a novel coronavirus vaccine is provided, the novel coronavirus vaccine comprising a nucleic acid molecule encoding a novel coronavirus S protein, wherein the amino acid sequence of the novel coronavirus S protein is selected from the S protein of the novel coronavirus Omeprion BA.5 sub-variant, the novel coronavirus Omeprion BA.2 sub-variant, the novel coronavirus Omeprion BA.3 sub-variant, or the novel coronavirus Omeprion BA.5 sub-variant.

[0008] In one or more embodiments, the amino acid sequence of the S protein of the novel coronavirus Omeprón variant BA.1 sub-variant is preferably as shown in SEQ ID. NO. 6, or contains an amino acid sequence that is at least 80% identical to that in SEQ ID. NO. 6, for example, but not limited to, an amino acid sequence that contains at least 80%, 85%, 90%, 95%, or 98% identical to that in SEQ ID. NO. 6.

[0009] In one or more embodiments, the amino acid sequence of the S protein of the novel coronavirus Omeprón variant BA.2 sub-variant is preferably as shown in SEQ ID. NO.4, or contains an amino acid sequence that is at least 80% identical to SEQ ID. NO.4, for example, but not limited to, an amino acid sequence that contains at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID. NO.4.

[0010] In one or more embodiments, the amino acid sequence of the S protein of the novel coronavirus Omeprón variant BA.3 sub-variant is preferably as shown in SEQ ID. NO.8, or contains an amino acid sequence that is at least 80% identical to SEQ ID. NO.8, for example, but not limited to, an amino acid sequence that contains at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID. NO.8.

[0011] In one or more embodiments, the amino acid sequence of the S protein of the novel coronavirus Omeprón variant BA.5 sub-variant is preferably as shown in SEQ ID. NO. 16 or contains an amino acid sequence that is at least 80% identical to SEQ ID. NO. 16, for example, it can be, but is not limited to, an amino acid sequence that contains at least 80%, 85%, 90%, 95% or 98% identical to SEQ ID. NO. 16.

[0012] In one or more embodiments, the amino acid sequence of the novel coronavirus S protein is selected from the S protein of the novel coronavirus Omeprón BA.5 sub-variant.

[0013] In one or more embodiments, the nucleic acid molecule includes DNA molecules and / or RNA molecules.

[0014] In one or more embodiments, the DNA molecule includes a stranded DNA molecule and / or a circular DNA molecule.

[0015] In one or more embodiments, the RNA molecule includes mRNA or circular RNA.

[0016] In one or more embodiments, the nucleic acid molecule is mRNA.

[0017] In one or more embodiments, the total GC% content of the open reading frame portion of the mRNA is 30% to 70%, and the GC% content of any 60bp fragment of the open reading frame is not less than 40%.

[0018] In one or more embodiments, the total GC% content of the open reading frame portion of the mRNA is 50% to 60%, more preferably 54% to 60%.

[0019] In one or more embodiments, the mRNA further includes one or more of the following: a 5' cap, a 5' UTR, a 3' UTR, a polyA tail, a start region, a stop region, a signal sequence region, and a linker sequence.

[0020] In one or more embodiments, the nucleotide sequence of the mRNA open reading frame is shown in SEQ ID.NO.17, SEQ ID.NO.19-22, SEQ ID.NO.10, SEQ ID.NO.11, SEQ ID.NO.12, SEQ ID.NO.13, SEQ ID.NO.14, SEQ ID.NO.15, and SEQ ID.NO.18.

[0021] In one or more embodiments, the nucleotide sequence of the mRNA open reading frame is shown in SEQ ID NO. 10–15 and SEQ ID NO. 17–18.

[0022] In one or more embodiments, the nucleotide sequence of the mRNA open reading frame is shown in SEQ ID NO. 17.

[0023] In an optional embodiment, based on the provided RNA sequence, those skilled in the art will be able to obtain the corresponding DNA sequence (e.g., uracil to thymine conversion). Similarly, based on the provided DNA sequence, those skilled in the art will obtain the corresponding RNA sequence (e.g., thymine to uracil conversion). In an optional embodiment, based on the provided RNA or DNA sequence, those skilled in the art will be able to obtain the corresponding amino acid sequence.

[0024] In one or more embodiments, the 5' cap is selected from ARCA, m7G(5"")ppp(5"")(2""OMeA)pG, m7G(5"")ppp(5"")(2""OMeG)pG, m7(3""OMeG)(5"")ppp(5"")(2""OMeG)pG, m7(3""OMeG)(5"")ppp(5"")(2""OMeA)pG, mCAP, dmCAP, tmCAP, or dmCAP.

[0025] In one or more embodiments, the length of the poly(A) is 50 to 200, preferably 80 to 200.

[0026] In one or more embodiments, the length of the 5'UTR is preferably 10 to 200 nucleotides, more preferably 15 to 100 nucleotides.

[0027] In one or more embodiments, the 5'UTR nucleotide sequence is as shown in SEQ ID.NO.1.

[0028] In one or more embodiments, the 3'UTR sequence is as shown in SEQ ID.NO.2.

[0029] In one or more embodiments, one or more uridines in the mRNA molecule are replaced with a modified nucleoside; preferably, the modified nucleoside replacing the uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0030] In one or more embodiments, the novel coronavirus vaccine contains nucleic acid lipid nanoparticles composed of the nucleic acid molecules and lipid components.

[0031] In one or more embodiments, the lipid component comprises, by molar percentage, 20-50% protonable cationic lipids, 20-50% structural lipids, 5-20% accessory lipids and 1-5% surfactants, wherein the total molar content of protonable cationic lipids, structural lipids, accessory lipids and surfactants is 100%.

[0032] In one or more embodiments, the protonable cationic lipid includes at least one of DlinMC3-DMA, DODMA, C12-200, and DlinDMA.

[0033] In one or more embodiments, the assisting lipids include at least one of DSPC, DOPE, DOPC, DOPG, and DOPS.

[0034] In one or more embodiments, the structural lipids include cholesterol and / or cholesterol derivatives.

[0035] In one or more embodiments, the surfactant includes at least one of PEG-DMG, PEG-DSPE, and TPGS.

[0036] In one or more embodiments, the lipid components, in molar percentage, comprise 50% Dlin-MC3-DMA, 10% DOPG, 38.5% cholesterol, and 1.5% PEG-DMG.

[0037] In one or more embodiments, the lipid composition comprises, in molar percentage, 50% Dlin-MC3-DMA, 20% DOPG, 29% cholesterol, and 1% PEG-DMG.

[0038] In one or more embodiments, the nucleic acid lipid nanoparticles were prepared according to the following method:

[0039] The aqueous phase containing nucleic acid molecules and the organic phase containing the lipid components are mixed evenly to obtain a mixture. After removing the organic phase, the concentration of nucleic acid molecules in the system is made to be 1-100 μg / ml to obtain the nucleic acid lipid nanoparticles.

[0040] The aqueous phase is an aqueous buffer containing 0.08–1.2 mg / L nucleic acid molecules, and the aqueous buffer is a citrate buffer or a sodium acetate buffer.

[0041] The organic phase is anhydrous C1-C4 low-carbon alcohol containing 5-7 mg / L of the lipid component; the volume ratio of the aqueous phase to the organic phase is 1:2-4.

[0042] In one or more embodiments, any of the aforementioned novel coronavirus vaccines is configured for use in products for the prevention or treatment of diseases caused by the novel coronavirus.

[0043] In one or more embodiments, a product configured to prevent or treat a disease caused by a novel coronavirus is characterized by comprising a novel coronavirus vaccine as described in any of the preceding embodiments.

[0044] Details of one or more embodiments of this disclosure are set forth in the following drawings and description. Other features, objects, and advantages of this disclosure will become apparent from the specification, drawings, and claims. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, reference may be made to one or more accompanying drawings. The appendices or examples used to describe the drawings should not be considered as limitations on the scope of any of the disclosed invention, the currently described embodiments and / or examples, or the best mode of these inventions as currently understood. The drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure, and those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0046] Figure 1 The results show the neutralizing activity of serum generated from C57 mice after immunizing them with the various vaccine formulations in Example 4 against the pseudovirus.

[0047] Figure 2 The results show the neutralizing activity of serum generated from C57 mice after immunizing them with the various vaccine formulations in Example 6 against the pseudovirus. Detailed Implementation

[0048] The technical solutions of this disclosure will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. Based on the embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0049] It should be noted that, unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions; all technical features and preferred features mentioned herein can be combined to form new technical solutions; and the components involved or their preferred components can be combined to form new technical solutions.

[0050] The “scope” disclosed in this disclosure is in the form of a lower limit and an upper limit, and may be one or more lower limits and one or more upper limits; unless otherwise stated, the operation steps may be performed sequentially or not in sequence.

[0051] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those familiar to a person skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to this disclosure.

[0052] According to various embodiments of this disclosure, a novel coronavirus vaccine is provided, which primarily uses nucleic acid molecules as immunogenic materials, including nucleic acid molecules encoding the BA.5S protein of the Omicron variant of the novel coronavirus. After immunization with this novel coronavirus vaccine, the BA.5S protein of the Omicron variant can be expressed in the body.

[0053] The term "nucleic acid molecule" as used in this disclosure refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acids include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA; vector DNA integrated with a foreign gene, such as an expression cassette or plasmid; DNA-RNA hybrids; or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0054] In the novel coronavirus vaccine provided in this disclosure, the S protein of the novel coronavirus encoded by the open reading frame may optionally be the S protein obtained by mutation of the Omeprone variant under natural conditions; or may optionally be the S protein obtained by artificial mutation and modification, wherein the mutation and modification may be to obtain an amino acid sequence of the S protein conforming to the Omeprone variant by mutation and modification of the wild type; or may be the amino acid sequence of the S protein obtained by further mutation and modification based on the amino acid sequence of the Omeprone variant S protein.

[0055] In one or more embodiments, the amino acid sequence of the S protein of the novel coronavirus encoded by the open reading frame is selected from the amino acid sequence of the S protein of the novel coronavirus Omecron variant, which is derived from the S protein of the BA.5 sub-variant, BA.2 sub-variant, BA.3 sub-variant, or the S protein of the BA.5 sub-variant; or, the amino acid sequence of the S protein of the Omecron variant may optionally be derived from the amino acid sequence obtained by mutation of the wild-type S protein.

[0056] Sequence identity between two nucleotide sequences indicates the percentage of identical nucleotides between the sequences. Sequence identity between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0057] The term "% identity" or similar term refers to the percentage of identical nucleotides or amino acids between sequences being compared at optimal alignment. This percentage is purely statistical, and the differences between the two sequences may (but are not necessarily) be randomly distributed across the entire length of the sequences being compared. Comparison of two sequences is typically performed after optimal alignment by comparing equivalent sequences relative to fragments or a "comparison window" to identify local regions of the corresponding sequences.

[0058] SEQ ID.NO.16 is the S protein of the Omicron BA.5 strain, which has been SN-mutated (with two proline residues substituted at positions 981 and 982 in the full-length S protein amino acid sequence), resulting in higher expression levels.

[0059] The amino acid sequence positions described in this disclosure are based on the full-length amino acid sequence of the wild-type S protein of the original strain of the novel coronavirus.

[0060] The novel coronavirus vaccine disclosed herein uses nucleic acid molecules as its main active ingredient. After administration to the body, this vaccine expresses and produces the S protein of the Omega strain of the virus. To further improve the vaccine's immunogenicity, this disclosure also optimizes the nucleic acid molecules and the vaccine formulation.

[0061] The open reading frame (ORF) described in this disclosure is a normal nucleotide sequence of a structural gene. The reading frame from the start codon to the stop codon encodes a complete polypeptide chain, and there is no stop codon in between that would interrupt translation.

[0062] In one or more embodiments, nucleic acid molecules can be sequence optimized using mRNA sequences to improve properties related to expression efficacy after in vivo administration. These improvements include: enhancing mRNA stability; increasing translational efficiency in target tissues; reducing the number of truncated proteins expressed; improving the folding of expressed proteins or preventing misfolding; reducing the toxicity of the expression product; reducing cell death induced by the expression product; and increasing and / or reducing protein aggregation, resulting in mRNAs with improved properties. Sequence optimization also aims to: optimize the formulation and delivery characteristics of nucleic acid-based therapeutics while maintaining structural and functional integrity; overcome expression thresholds; increase expression rates; improve half-life and / or protein concentration; optimize protein localization; and avoid adverse biological responses such as immune responses and / or degradation pathways. Sequence optimization techniques include: (1) codon optimization based on codon frequencies in specific organs and / or host organisms to ensure proper folding and expression; (2) adjusting G / C content to increase mRNA stability or reduce secondary structures; (3) minimizing tandem repeat codons or base runs that may impair gene construction or expression; (4) customizing transcription and translation control regions; and (5) reducing or eliminating problematic secondary structures within polynucleotides.

[0063] Figure 1 and Figure 2 The diagram shows the EC50 values ​​of the original strain of the SARS-CoV-2 pseudovirus, labeled "Original Strain," "Alpha," "Beta," "Gamma," "Delta," "BA.1," "BA.2," and "BA.4 & BA.5" (the S protein of the BA.4 & BA.5 sub-variants is the same).

[0064] The technical solutions and beneficial effects of this disclosure are further illustrated below with reference to preferred embodiments.

[0065] Example 1

[0066] This embodiment provides a method for preparing lipid nanoparticles containing RNA, wherein the lipid nanoparticles comprise, by molar percentage: 50% Dlin-MC3-DMA, 20% DOPG, 29% cholesterol, and 1% PEG-DMG. The preparation method is as follows:

[0067] (a) Dissolve RNA in citrate buffer at pH 4 and adjust the concentration to 0.1 mg / mL to obtain the aqueous phase. (b) Dissolve Dlin-MC3-DMA, DOPG, cholesterol, and PEG-DMG in anhydrous ethanol according to the formulation amounts, and adjust the concentration of lipid components in the organic phase to 6 mg / mL to obtain the organic phase.

[0068] (c) The aqueous phase from step (a) and the organic phase from step (b) were mixed at a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 mL / min. The mixture was immediately diluted 100 times with PBS solution at pH 7.4, and ethanol was removed from the solution using tangential flow filtration (TFF). The mixture was then concentrated to a concentration of 55 μg / ml of mRNA to obtain lipid nanoparticles containing RNA encoding SARS-CoV-2 viral antigen.

[0069] Example 2

[0070] Using luciferase as a reporter gene, the efficiency of different vaccine vector formulations (as shown in the table below, "MC3" refers to Dlin-MC3-DMA, "+" indicates that luciferase expression in mice was detected by a small animal in vivo fluorescence imaging system after administration) in delivering mRNA encoding the luciferase gene in mice was studied using in vivo fluorescence imaging technology. The physicochemical properties of different compound formulations (preparation method see Example 1) were also tested, and the results are shown in the table.

[0071] Table 1

[0072] .

[0073] Studies have shown that increasing the lipid-to-mRNA mass ratio improves the encapsulation efficiency of mRNA in lipid nanoparticles, thereby enhancing its stability. Furthermore, moderately increasing the polyethylene glycol (PEG) content in the formulation is beneficial for improving mRNA expression efficiency in vivo. Therefore, considering factors such as mRNA encapsulation efficiency and in vivo mRNA delivery efficiency, formulations 3 and 4 were selected for subsequent mRNA vaccine research.

[0074] Example 3

[0075] Table 2 shows the ability of different formulations of cationic lipid nanoparticles to encapsulate the full-length mRNA encoding the S protein and the particle size data of the formed nanoparticles. All formulations can compress the S protein mRNA into nanoparticles with a particle size of less than 100 nm and a net neutral surface potential, while also encapsulating at least 50% of the mRNA, thus all possessing a certain in vivo delivery effect. "MC3" refers to Dlin-MC3-DMA.

[0076] Table 2

[0077] .

[0078] Example 4

[0079] Screening of the S protein of Omicron strain subtypes:

[0080] This embodiment designs a series of mRNA sequence information, among which the open reading frame sequences are shown in Table 3; in addition to the open reading frame sequences, the characteristics of this series of mRNA sequences also include a 5' cap (m7G(5')(2'-OMeA)pG), a 5' UTR (as shown in SEQ ID.NO.1), a 3' UTR (as shown in SEQ ID.NO.2), and a 3' tail with 100 polyA (as shown in SEQ ID.NO.3).

[0081] Table 3 mRNA sequence design scheme

[0082] .

[0083] Four different antigens, samples 1-4, were prepared into vaccines according to the method in Example 1. The mRNA mixtures of different groups were prepared into LNP preparations. The encapsulation rate of the LNP preparations was tested to be over 90%, and the particle size was about 70 nm. Sample 5 in the figure is a blank control (placebo).

[0084] The prepared mRNA LNP formulation was used in an immunization experiment in C57 mice. Each mouse received a 5 μg injection (based on mRNA) via intramuscular injection into the lateral thigh of the hind limb. A second immunization was administered 7 days later, with 3 mice per group. Serum was collected from mice 14 days after the first immunization and sent to a third-party laboratory for SARS-CoV-2 pseudovirus neutralization activity testing. Mouse serum was diluted at different ratios (initial dilution factor 30) in 96-well plates, and SARS-CoV-2 infective pseudoviruses were added. Cell controls and virus controls were also included. After incubation for 1 hour, pre-prepared cells were added and cultured in a cell culture incubator for 20–28 hours. A portion of the supernatant was discarded, and luciferase assay reagent was added. After reaction at room temperature in the dark, the cells were repeatedly pipetted and aspirated to ensure complete cell lysis. The cells were then placed in a chemiluminescence analyzer to read the luminescence value. The Reed-Muench method was used to calculate the EC50 value, ensuring the validity of the virus and cell controls. The EC50 values ​​of the pseudovirus strains with different antigens prepared for samples 1–4 are shown in the figure. Figure 1 As shown.

[0085] from Figure 1 The results show that among vaccine formulations prepared with open reading frames of mRNA vaccines encoding different S proteins (amino acid sequences as shown in SEQ ID. NO. 4, SEQ ID. NO. 6, SEQ ID. NO. 8, and SEQ ID. NO. 16), the mRNA vaccine encoding the S protein with an amino acid sequence as shown in SEQ ID. NO. 16 (encoding the S protein of the SARS-CoV-2 Omicron BA.5 strain) showed less reduction in the activity of neutralizing antibodies against various sub-variants of SARS-CoV-2 Omicron in mice.

[0086] Example 5

[0087] Optimization of the mRNA sequence encoding the S protein of the BA.5 sub-mutant strain:

[0088] Based on the optimization principles of the S protein (amino acid sequence shown in SEQ ID NO. 16) and mRNA sequence of the Omicron strain of SARS-CoV-2, a series of mRNA sequences were designed in this embodiment. The open reading frame information is shown in Table 4 below.

[0089] In addition to the open reading frames in Table 4, the mRNA sequence features of this series also include a 5' cap (m7G(5')(2'-OMeA)pG), a 5' UTR (as shown in SEQ ID.NO.1), a 3' UTR (as shown in SEQ ID.NO.2), and a 3' tail with 100 polyA (as shown in SEQ ID.NO.3).

[0090] Table 4. mRNA sequence design scheme and relative expression level

[0091] .

[0092] Table 4 shows the "local GC% content": the GC% content in a local sequence with a window size of 60bp, from the 3' end to the 5' end of the ORF.

[0093] Cells were transfected with the mRNAs shown in Table 4, and the expression of the full-length S protein in the cells was detected. The results are shown in Table 4. The detailed method is as follows: HEK293 cells transfected with each mRNA for 24 hours were lysed. At a sample loading rate of 10 μg total protein, SDS-PAGE immunoblotting was used to specifically detect the target protein. In this embodiment, anti-SARS-S1 protein antibody was used as the primary antibody, and goat anti-mouse-HRP antibody was used as the secondary antibody for incubation, followed by color development. When analyzing the protein expression levels, β-actin was used as an internal control for standardized quantification. Cells not transfected with mRNA were set up as a negative control to compare the differences in protein expression levels after cell transfection with different mRNAs. The results showed that the expression of the full-length S protein and the S1 subunit could be detected in all cases. The expression levels of each sequence were measured as relative OD values, as shown in Table 4; the relative OD value was calculated as: sample OD value / OD value of sample 1.

[0094] As shown in Table 4, when the overall GC% content of the mRNA open reading frame sequence is 54-60%, and the local GC% content is not less than 40%, the relative expression level of the S protein is relatively high (see SEQ ID. NO. 17 and 19). In addition, compared with the mRNA open reading frame sequence SEQ ID. NO. 19, the S protein expression level of the mRNA open reading frame sequence SEQ ID. NO. 17 is higher.

[0095] Samples 1 and 2 from Example 5, containing two different antigens, were prepared into vaccines according to the method in Example 1. The mRNA mixtures from different groups were then prepared into LNP formulations. The encapsulation efficiency of the LNP formulations was found to be above 90%, with a particle size of approximately 70 nm. The two vaccine formulations were used in C57 mouse immunization experiments, and the experimental methods are shown in Example 4. The corresponding serum group numbers and results are as follows. Figure 2 As shown.

[0096] from Figure 2 As can be seen, the mRNA vaccine preparation made from the mRNA sequence SEQ ID. NO.17 exhibits the best antibody neutralization capacity due to the high expression level of the mRNA sequence SEQ ID. NO.17.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this disclosure, and are not intended to limit them. Although this disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this disclosure.

Claims

1. A novel coronavirus vaccine, characterized in that, The novel coronavirus vaccine contains nucleic acid lipid nanoparticles composed of nucleic acid molecules encoding the novel coronavirus S protein and lipid components; the novel coronavirus S protein is selected from the S protein of the novel coronavirus Omeprone BA.5 sub-variant strain as shown in SEQ ID NO:

16. The nucleic acid molecule is mRNA, and the nucleotide sequence of the open reading frame of the mRNA is shown in SEQ ID NO:17; The lipid composition comprises, by molar percentage, 50% Dlin-MC3-DMA, 10% DOPG, 38.5% cholesterol, and 1.5% PEG-DMG, or, by molar percentage, the lipid composition comprises 50% Dlin-MC3-DMA, 20% DOPG, 29% cholesterol, and 1% PEG-DMG.

2. The novel coronavirus vaccine according to claim 1, characterized in that, The nucleic acid lipid nanoparticles were prepared by the following method: an aqueous phase containing nucleic acid molecules and an organic phase containing the lipid components were mixed evenly to obtain a mixture. After removing the organic phase, the concentration of nucleic acid molecules in the system was adjusted to 1–100 μg / ml to obtain the nucleic acid lipid nanoparticles. The aqueous phase was an aqueous buffer containing 0.08–1.2 mg / L of nucleic acid molecules, and the aqueous buffer was citrate buffer or sodium acetate buffer. The organic phase was anhydrous C1–C4 low-carbon alcohol containing 5–7 mg / L of the lipid components. The volume ratio of the aqueous phase to the organic phase was 1:2–4.

3. The use of the novel coronavirus vaccine according to any one of claims 1-2 in the preparation of a product configured to prevent the novel coronavirus.

4. A product configured to prevent the novel coronavirus, characterized in that, It includes the novel coronavirus vaccine as described in any one of claims 1-2.

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