A novel coronavirus vaccine and a preparation method thereof

Abstract

The application discloses a novel coronavirus vaccine and a preparation method thereof, and relates to the technical field of immunity. The application is characterized in that the sequence of the coding region of mRNA is optimized and adjusted according to the characteristics of the mutant strain of the novel coronavirus, and the optimized mRNA can deliver the S protein with strong immunogenicity in the body, so that the antibodies generated by the body have a neutralizing effect on the mutant strain of the novel coronavirus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology, in particular, to a COVID-19 vaccine and a preparation method thereof. BACKGROUND

[0002] Corona Virus Disease 2019 (COVID-19, also known as novel coronavirus) is a novel coronavirus of the beta genus. Currently, vaccines that have made considerable research progress in the field of vaccine treatment include polypeptide vaccines, nucleic acid vaccines, and viral vector vaccines, etc.

[0003] Nucleic acid vaccines include RNA vaccines and DNA vaccines. Among them, RNA vaccines are a class of mRNA sequences encoding pathogen-specific proteins (antigens), which are usually encapsulated in lipid nanoparticles to prevent degradation and in vivo delivery. Once the vaccine sequence is expressed in vivo, the target antigen is recognized by the immune system, thereby inducing the immune response of the body.

[0004] As an RNA virus, the coronavirus is highly variable. In the first half of 2020, the main epidemic strain was the original strain, and the currently marketed and most of the COVID-19 vaccines in the clinical trial stage were designed for antigens against this strain. Since July 2020, the Alpha strain, Beta strain, Delta strain, and Omicron strain of the coronavirus have emerged in succession. Studies have found that the protective effect of existing vaccines against variant strains has decreased to varying degrees, especially against sub-variant strains of the Omicron strain. Therefore, a COVID-19 vaccine with better protection against variant strains is urgently needed. SUMMARY

[0005] In some embodiments, the present disclosure provides a COVID-19 vaccine comprising or encoding a COVID-19 antigen, which can induce an effective neutralizing antibody response against COVID-19.

[0006] Some aspects of the present disclosure provide a COVID-19 vaccine comprising or encoding a COVID-19 antigen, which is selected from a COVID-19 nucleic acid vaccine, a COVID-19 polypeptide vaccine, or a COVID-19 vaccine.

[0007] Some aspects of the present disclosure provide a COVID-19 vaccine comprising or encoding a COVID-19 antigen, which is a COVID-19 XBB.1.5 variant S protein; in some embodiments, the COVID-19 XBB.1.5 variant S protein includes at least one of a F813P substitution, an A888P substitution, an A895P substitution, an A938P substitution, a K982P substitution, and a V983P substitution.

[0008] In some embodiments, the S protein of the new coronavirus XBB.1.5 variant comprises a K982P substitution and a V983P substitution.

[0009] In some embodiments, the S protein of the new coronavirus XBB.1.5 variant has an amino acid sequence as set forth in Seq ID NO. 1.

[0010] In some embodiments, the new coronavirus vaccine is a new coronavirus nucleic acid vaccine.

[0011] In some embodiments, the new coronavirus nucleic acid vaccine comprises a nucleic acid molecule.

[0012] In some embodiments, the RNA molecule comprises an mRNA or a circular RNA.

[0013] In some embodiments, the mRNA molecule comprises an open reading frame (ORF) having a nucleotide sequence as set forth in Seq ID NO. 2-10.

[0014] In some embodiments, the mRNA further comprises at least one of a 5’ cap, a 5’ UTR, a 3’ UTR, and a 3’ polyA tail. In some embodiments, the 5’ cap is selected from ARCA, mCAP, dmCAP, tmCAP, m7G(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, dmCAP, or m7G(5')ppp(5')(2'OMeG)pG.

[0015] In some embodiments, the 3’ polyA tail has a length of 50-200.

[0016] In some embodiments, the 5’ UTR nucleotide sequence is as set forth in Seq ID NO. 11-13. In some embodiments, the 3’ UTR sequence is as set forth in Seq ID NO. 14-16.

[0017] In some embodiments, one or more uridines in the mRNA are replaced with modified nucleosides.

[0018] In some embodiments, the new coronavirus vaccine comprises a delivery formulation; in some embodiments, the delivery formulation comprises a lipid nanoparticle or a cationic liposome.

[0019] In some embodiments, the mRNA and the delivery formulation are mixed to form a new coronavirus vaccine; the delivery formulation comprises a lipid nanoparticle or a cationic liposome.

[0020] In some embodiments, the present disclosure provides an isolated mRNA, a coding region nucleic acid sequence of the mRNA encodes a S protein of a XBB.1.5 variant of a new coronavirus, the S protein of the XBB.1.5 variant of the new coronavirus has at least one of the following substitutions in the amino acid sequence: F813P, A888P, A895P, A938P, K982P, V983P.

[0021] In some embodiments, the present disclosure provides an isolated DNA, the DNA is obtained by reverse transcription of the nucleic acid of the isolated mRNA, or the DNA is a sequence capable of transcribing the isolated mRNA.

[0022] In some embodiments, the present disclosure provides a biological material, the biological material comprises any one of an expression cassette, a vector, an engineered bacterium, or a cell line, the biological material contains or expresses the isolated mRNA or the isolated DNA. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present disclosure, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0024] Figure 1 EC50 values for XBB.1.5 pseudovirus strain in Example 5.

[0025] Figure 2 EC50 values for XBB.1.16 pseudovirus strain in Example 5.

[0026] Figure 3 EC50 values for pseudovirus strain in Example 6.

[0027] Figure 4 Neutralization activity detection results for live virus strain in Example 7.

[0028] Figure 5 Western blot results of protein expression after DNA transfection of cells in Example 8. DETAILED DESCRIPTION

[0029] Some aspects of the present disclosure provide a coronavirus vaccine comprising or encoding a coronavirus antigen, the coronavirus vaccine selected from a coronavirus nucleic acid vaccine, a coronavirus polypeptide vaccine, or a coronavirus vaccine, the antigen being a coronavirus XBB.1.5 variant S protein.

[0030] In some embodiments, the coronavirus XBB.1.5 variant S protein comprises at least one of F813P substitution, A888P substitution, A895P substitution, A938P substitution, K982P substitution, V983P substitution.

[0031] In some embodiments, the coronavirus XBB.1.5 variant S protein comprises K982P substitution and V983P substitution.

[0032] The positions of the amino acid sequences described in the present disclosure, such as F813P substitution, A888P substitution, A895P substitution, A938P substitution, K982P substitution, V983P, are positioned based on the wild type full-length amino acid sequence of the coronavirus Omicron XBB.1.5 subvariant S protein (such as Seq ID NO. 1). For example, the K982P substitution is replacing the K amino acid residue at the 982th position of the wild type full-length amino acid sequence of the coronavirus Omicron XBB.1.5 subvariant S protein with a P amino acid residue based on the wild type of the coronavirus Omicron XBB.1.5 subvariant S protein.

[0033] In some embodiments, the amino acid sequence of the coronavirus Omicron XBB.1.5 subvariant S protein is set forth in Seq ID NO. 1, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 1, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 1.

[0034] In some embodiments, the coronavirus vaccine is a coronavirus nucleic acid vaccine. In some embodiments, the coronavirus nucleic acid vaccine comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises a DNA molecule and / or an RNA molecule. In some embodiments, the DNA molecule comprises a linear DNA molecule and / or a circular DNA molecule. In some embodiments, the RNA molecule comprises an mRNA or a circular RNA.

[0035] In some embodiments, the mRNA molecule comprises an open reading frame (ORF) having a nucleotide sequence as set forth in Seq ID NO. 2-10, or a nucleotide sequence comprising at least 80% identity to Seq ID NO. 2-10, for example, but not limited to, a nucleotide sequence comprising at least 80%, 85%, 90%, 95%, or 98% identity to Seq ID NO. 2-10.

[0036] In some embodiments, the nucleic acid molecule can optimize the mRNA sequence by sequence optimization means to improve properties related to expression efficacy after in vivo administration: for example, to improve mRNA stability, to increase translation efficacy in target tissues, to reduce the amount of truncated proteins expressed, to improve folding or prevent misfolding of the expressed protein, to reduce toxicity of the expression product, to reduce cell death caused by the expression product, to increase and / or reduce protein aggregation, to result in an mRNA with improved properties. The purpose of sequence optimization also includes: optimizing the formulation and delivery of nucleic acid-based therapeutics while maintaining structural and functional integrity; overcoming thresholds of expression; increasing expression rates; half-life and / or protein concentration; optimizing protein localization; and avoiding adverse biological responses such as immune responses and / or degradation pathways. Sequence optimization means include: (1) codon optimization according to codon frequencies in a particular organ and / or host organism to ensure proper folding and proper expression; (2) modulation of G / C content to increase mRNA stability or reduce secondary structures; (3) minimization of tandem repeated codons or base runs that can impair gene construction or expression; (4) customization of transcription and translation control regions; (5) reduction or elimination of problematic secondary structures within the polynucleotide.

[0037] “Sequence identity” between two nucleotide sequences indicates the percentage of nucleotides that are the same between the sequences. “Sequence identity” between two amino acid sequences indicates the percentage of amino acids that are the same between the sequences.

[0038] The term “% identity” or like terms refers to the percentage of nucleotides or amino acids that are the same between the sequences being compared, after optimal alignment. The percentage is purely statistical and the differences between the two sequences can be, but are not necessarily, randomly distributed along the entire length of the sequences being compared. Comparison of two sequences is typically performed by comparing the sequences to each other after optimal alignment, over a segment or “comparison window” to identify local regions of sequence correspondence.

[0039] In some embodiments, based on the provided mRNA open reading frame sequence, one of ordinary skill in the art would be able to obtain the corresponding circular RNA open reading frame sequence, and prepare the circular RNA complete sequence capable of encoding the same amino acid sequence according to the content disclosed in CN202180048567.4 and the like. In some embodiments, based on the provided mRNA sequence, one of ordinary skill in the art would be able to obtain the corresponding DNA sequence (e.g., uracil converted to thymine). Likewise, based on the provided DNA sequence, one of ordinary skill in the art would obtain the corresponding RNA sequence (e.g., thymine converted to uracil). In some embodiments, based on the provided RNA or DNA sequence, one of ordinary skill in the art would be able to obtain the corresponding amino acid sequence.

[0040] In some embodiments, the mRNA molecule comprises a self-replicating mRNA molecule or a non-self-replicating mRNA molecule.

[0041] In some embodiments, the mRNA described in the present disclosure is a self-replicating mRNA carrying a sequence capable of expressing a RNA polymerase (RNA-dependent RNA polymerase, RdRP), and the sequence design content of the self-replicating mRNA in Chinese patent CN202110424124.2 is incorporated into the present disclosure.

[0042] In some embodiments, the mRNA is a non-self-replicating mRNA.

[0043] In some embodiments, the mRNA further comprises at least one of a 5' cap, a 5' UTR, a 3' UTR, and a 3' polyA tail.

[0044] The 5' untranslated region (UTR) described in the present disclosure refers to the sequence of the mRNA that does not encode a polypeptide located immediately upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript for ribosomal translation). When the RNA transcript is produced, the 5' UTR can comprise a promoter sequence. Such promoter sequences are known in the art. The RNA sequence of the 5' UTR is shown in one of Seq ID NO. 11, Seq ID NO. 12, or Seq ID NO. 13, or the 5' UTR sequence disclosed before the filing date of the present disclosure can also be introduced into the present disclosure.

[0045] The 3' untranslated region (UTR) described in the present disclosure refers to the sequence of the mRNA that does not encode a polypeptide located downstream of the stop codon. The RNA sequence of the 3' UTR is shown in one of Seq ID NO. 14, Seq ID NO. 15, or Seq ID NO. 16, or the 3' UTR sequence disclosed before the filing date of the present disclosure can also be introduced into the present disclosure.

[0046] The poly(A) tail described in the present disclosure is a sequence downstream of the 3' UTR containing multiple consecutive adenosine monophosphates of an mRNA. The poly(A) tail can contain 10 to 300 adenosine monophosphates. In cells and / or in vivo, the poly(A) tail serves to protect the mRNA from enzymatic degradation, and aids in transcription termination, and / or mRNA export from the nucleus and translation.

[0047] In some embodiments, the 5' cap is selected from ARCA, mCAP, dmCAP, tmCAP, m7G(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, dmCAP, or m7G(5')ppp(5')(2'OMeG)pG.

[0048] In some embodiments, the 5' cap is m7Gppp(5')(2'-OMeA)pG.

[0049] In some embodiments, the 3' polyA tail has a length of 50-200. In some embodiments, the 3' polyA tail has a length of 80-200. In some embodiments, the 3' polyA tail has a length of 80-120. In some embodiments, the 3' polyA tail has a length of 120.

[0050] In some embodiments, the 5' UTR has a length of 10-200 nucleotides. In some embodiments, the 5' UTR has a length of 15-100 nucleotides. In some embodiments, the 5' UTR nucleotide sequence is as set forth in Seq ID NO. 11-13. In some embodiments, the 5' UTR nucleotide sequence is as set forth in Seq ID NO. 11.

[0051] In some embodiments, the 3' UTR sequence is as set forth in Seq ID NO. 14-16. In some embodiments, the 3' UTR sequence is as set forth in Seq ID NO. 14.

[0052] In some embodiments, one or more uridines in the mRNA are replaced with a modified nucleoside.

[0053] In some embodiments, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0054] In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ). In some embodiments, the 5' cap is m7Gppp(5')(2'-OMeA)pG.

[0055] In some embodiments, the coronavirus vaccine comprises a delivery formulation; in some embodiments, the delivery formulation comprises a lipid nanoparticle or a cationic liposome.

[0056] In some embodiments, the mRNA and the delivery formulation are mixed to form a coronavirus vaccine; the delivery formulation comprises a lipid nanoparticle or a cationic liposome.

[0057] In some embodiments, the components of the lipid nanoparticle comprise at least one of a protonatable cationic lipid, a structural lipid, a helper lipid, and a surfactant.

[0058] In some embodiments, the components of the lipid nanoparticle comprise, by weight fraction, 40-60 parts of a protonatable cationic lipid, 20-40 parts of a structural lipid, 10-30 parts of a helper lipid, and 0.5-5 parts of a surfactant.

[0059] In some embodiments, the protonatable cationic lipid is selected from at least one of Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA.

[0060] In some embodiments, the structural lipid comprises cholesterol and / or a cholesterol derivative.

[0061] In some embodiments, the helper lipid comprises at least one of DSPC, DOPE, DOPC, DOPG, and DOPS.

[0062] In some embodiments, the surfactant comprises at least one of PEG-DMG, PEG-DSPE, and TPGS.

[0063] In some embodiments, the liposome nanoparticle comprises, by mole percent, 20-50% of a cationic lipid, such as, but not limited to, 20%, 25%, 30%, 35%, 40%, 45%, or 50%; 20-50% of DOPG, such as, but not limited to, 20%, 25%, 30%, 35%, 40%, 45%, or 50%; 5-20% of cholesterol, such as, but not limited to, 5%, 10%, 15%, or 20%; and 1-5% of PEG-DMG, such as, but not limited to, 1%, 2%, 3%, 4%, or 5%.

[0064] In some embodiments, the liposome nanoparticle comprises 20-50% cationic lipid by mole percentage, for example, but not limited to, 20%, 25%, 30%, 35%, 40%, 45%, or 50%; 20-50% DSCP by mole percentage, for example, but not limited to, 20%, 25%, 30%, 35%, 40%, 45%, or 50%; 5-20% cholesterol by mole percentage, for example, but not limited to, 5%, 10%, 15%, or 20%; and 1-5% PEG-DMG by mole percentage, for example, but not limited to, 1%, 2%, 3%, 4%, or 5%.

[0065] In some embodiments, the liposome nanoparticle comprises 50% Dlin-MC3-DMA, 10% DOPG, 38.5% cholesterol, and 1.5% PEG-DMG by mole percentage.

[0066] In some embodiments, the liposome nanoparticle comprises 50% Dlin-MC3-DMA, 10% DSCP, 38.5% cholesterol, and 1.5% PEG-DMG by mole percentage.

[0067] In some embodiments, the present disclosure provides a method for preparing a COVID-19 vaccine according to any of the preceding embodiments, characterized in that it comprises: mixing the mRNA and a delivery formulation to form a COVID-19 vaccine; the delivery formulation comprises a lipid nanoparticle or a cationic liposome.

[0068] In some embodiments, the method for preparing a COVID-19 vaccine, the mRNA is dissolved in a buffer to obtain an aqueous phase, and each lipid component of the liposome nanoparticle is dissolved in an organic solvent to obtain an organic phase, and the aqueous phase and the organic phase are mixed to remove the organic phase to obtain a COVID-19 vaccine.

[0069] In some embodiments, the volume ratio of the aqueous phase to the organic phase is 1:2-4, preferably 1:3.

[0070] In some embodiments, the buffer comprises a citrate buffer or sodium acetate, preferably a citrate buffer.

[0071] In some embodiments, the pH of the buffer is 3-7, preferably 4.

[0072] In some embodiments, the concentration of mRNA in the aqueous phase is 0.05-0.5 mg / mL, preferably 0.1 mg / mL.

[0073] In some embodiments, the organic solvent is selected from C1-C4 low-carbon alcohol, preferably anhydrous ethanol.

[0074] In some embodiments, the concentration of the lipid component in the organic phase is 5 mg / mL to 7 mg / mL, preferably 6 mg / mL.

[0075] In some embodiments, the microfluidic mixing is used to mix the aqueous phase and the organic phase, and the organic solvent is filtered using tangential flow filtration;

[0076] Preferably, the flow rate of the microfluidic is > 3 ml / min, further preferably 12 mL / min.

[0077] In some embodiments, after mixing, a concentration step is further included, which makes the final concentration of the mRNA 50 pg / mL to 200 pg / mL, preferably 100 pg / mL.

[0078] In some embodiments, the diameter of the lipid nanoparticle is less than about 200 nm. In some embodiments, the diameter of the lipid nanoparticle is less than about 150 nm. In some embodiments, the diameter of the lipid nanoparticle is less than 100 nm. In some embodiments, the diameter of the lipid nanoparticle is about 55 nm to about 90 nm.

[0079] In some embodiments, the coronavirus vaccine is a coronavirus polypeptide vaccine. The coronavirus polypeptide vaccine is a coronavirus vaccine prepared by chemical synthesis technology or genetic engineering technology according to the amino acid sequence of a known or predicted antigen epitope in the S protein gene of the coronavirus Omicron XBB.1.5 sub-variant. In some embodiments, the coronavirus polypeptide vaccine described in the present disclosure is a coronavirus vaccine prepared by chemical synthesis technology according to the amino acid sequence of the S protein of the coronavirus XBB.1.5 variant, combined with artificial mutations and modifications. In some embodiments, the coronavirus polypeptide vaccine described in the present disclosure is a coronavirus vaccine obtained after fermentation by genetic engineering technology (such as by constructing a genetically engineered bacterium through genetic engineering technology) according to the amino acid sequence of the S protein of the coronavirus XBB.1.5 variant, combined with artificial mutations and modifications. In some embodiments, the coronavirus polypeptide vaccine comprises a polypeptide antigen amino acid sequence as shown in Seq ID NO. 1.

[0080] In some embodiments, the coronavirus vaccine described in the present disclosure carries the S protein gene of the coronavirus Omicron XBB.1.5 sub-variant into the human body through harmless microorganisms, inducing the immune system of the body to make an immune response. Important viruses used in the cell immunity test include variants of the bovine disease virus, poliovirus, etc. In some embodiments, the coronavirus vaccine described in the present disclosure comprises a nucleotide sequence encoding an antigen with an amino acid sequence as shown in Seq ID NO. 1.

[0081] In some embodiments, the present disclosure provides an isolated mRNA, wherein the coding region nucleic acid sequence of the mRNA encodes a S protein of a XBB.1.5 variant of a novel coronavirus, wherein the S protein of the XBB.1.5 variant of the novel coronavirus comprises at least one of the following substitutions in the amino acid sequence: F813P, A888P, A895P, A938P, K982P, V983P.

[0082] In some embodiments, the present disclosure provides the isolated mRNA, wherein the S protein of the XBB.1.5 variant of the novel coronavirus comprises the K982P substitution and the V983P substitution.

[0083] In some embodiments, the present disclosure provides the isolated mRNA, wherein the S protein of the XBB.1.5 variant of the novel coronavirus has an amino acid sequence as set forth in Seq ID NO. 1, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 1, such as, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 1.

[0084] In some embodiments, the present disclosure provides the isolated mRNA, wherein the mRNA molecule comprises an open reading frame (ORF) having a nucleotide sequence as set forth in Seq ID NO. 2-10, or comprises a nucleotide sequence that is at least 80% identical to Seq ID NO. 2-10, such as, but not limited to, a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 2-10.

[0085] In some embodiments, the present disclosure provides the isolated mRNA, wherein based on the provided mRNA open reading frame sequence, a person of ordinary skill in the art would be able to obtain the corresponding circular RNA open reading frame sequence, and prepare a circular RNA complete sequence capable of encoding the same amino acid sequence according to the content of CN202180048567.4 and the like.

[0086] In some embodiments, the present disclosure provides the isolated mRNA, wherein the mRNA molecule comprises a self-replicating mRNA molecule or a non-self-replicating mRNA molecule.

[0087] In some embodiments, the present disclosure provides the isolated mRNA, wherein the mRNA described in the present disclosure is a self-replicating mRNA carrying a sequence capable of expressing a RNA polymerase (RNA-dependent RNA polymerase, RdRP), and the sequence design content of the self-replicating mRNA of CN202110424124.2 is incorporated into the present disclosure.

[0088] In some embodiments, the present disclosure provides the isolated mRNA, wherein the 5' cap is selected from ARCA, mCAP, dmCAP, tmCAP, m7G(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, dmCAP, or m7G(5')ppp(5')(2'OMeG)pG.

[0089] In some embodiments, the present disclosure provides the isolated mRNA, wherein the 5' cap is selected from ARCA, mCAP, dmCAP, tmCAP, m7G(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, dmCAP, or m7G(5')ppp(5')(2'OMeG)pG.

[0090] In some embodiments, the present disclosure provides the isolated mRNA, wherein the 5' cap is m7Gppp(5')(2'-OMeA)pG.

[0091] In some embodiments, the present disclosure provides the isolated mRNA, wherein the 3' polyA tail has a length of 50-200.

[0092] In some embodiments, the present disclosure provides the isolated mRNA, wherein the 3' polyA tail has a length of 80-200.

[0093] In some embodiments, the present disclosure provides the isolated mRNA, wherein the 3' polyA tail has a length of 80-120. In some embodiments, the 3' polyA tail has a length of 120.

[0094] In some embodiments, the present disclosure provides the isolated mRNA, wherein the 5' UTR has a length of 10-200 nucleotides.

[0095] In some embodiments, the present disclosure provides the isolated mRNA, wherein the 5' UTR has a length of 15-100 nucleotides.

[0096] In some embodiments, the present disclosure provides the isolated mRNA, wherein the 5' UTR nucleotide sequence is as set forth in Seq ID NO. 11-13.

[0097] In some embodiments, the present disclosure provides the isolated mRNA, wherein the 5' UTR nucleotide sequence is as set forth in Seq ID NO. 11.

[0098] In some embodiments, the present disclosure provides the isolated mRNA, wherein the 3' UTR sequence is as set forth in Seq ID NO. 14-16.

[0099] In some embodiments, the present disclosure provides the isolated mRNA, the 3’ UTR sequence is set forth in Seq ID NO. 14.

[0100] In some embodiments, the present disclosure provides the isolated mRNA, one or more uridines in the mRNA are replaced with modified nucleosides.

[0101] In some embodiments, the present disclosure provides the isolated mRNA, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0102] In some embodiments, the present disclosure provides the isolated mRNA, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0103] In some embodiments, the present disclosure provides an isolated DNA, the DNA is obtained by reverse transcription of the isolated mRNA nucleic acid, or the DNA is a sequence capable of transcribing the isolated mRNA.

[0104] In some embodiments, the present disclosure provides a biological material, the biological material comprises any one of an expression cassette, a vector, an engineered bacteria, or a cell line, the biological material contains or expresses the isolated mRNA or the isolated DNA.

[0105] In some embodiments, the present disclosure provides a method, the method comprises administering to a subject an effective amount of at least one of the SARS-CoV-2 vaccine of any one of the preceding embodiments, the isolated mRNA of any one of the preceding embodiments, the isolated DNA of any one of the preceding embodiments, or the biological material of any one of the preceding embodiments, to induce a neutralizing antibody response against SARS-CoV-2 in the subject.

[0106] In some embodiments, the method comprises administering to a subject at least one effective amount of at least one of the SARS-CoV-2 vaccine of any one of the preceding embodiments, the isolated mRNA of any one of the preceding embodiments, the isolated DNA of any one of the preceding embodiments, or the biological material of any one of the preceding embodiments.

[0107] In some embodiments, the effective amount of the present disclosure is as low as 40 pg, 30 pg, 25 pg, 20 pg, 15 pg, 10 pg, 5 pg, 3 pg, or 1 pg of the SARS-CoV-2 vaccine of any one of the preceding embodiments, the isolated mRNA of any one of the preceding embodiments, the isolated DNA of any one of the preceding embodiments, or the biological material of any one of the preceding embodiments.

[0108] Example 1: Process for RNA preparation of LNP

[0109] A lipid nanoparticle comprising RNA encoding a respiratory syncytial virus antigen, wherein the lipid nanoparticle comprises Dlin-MC3-DMA 50%, DOPG 20%, cholesterol 29%, and PEG-DMG 1% by mole percent.

[0110] The preparation method is as follows:

[0111] (a) Dissolve the RNA in a citrate buffer at pH 4, adjust the concentration to 0.1 mg / ml to obtain an aqueous phase.

[0112] (b) Dissolve Dlin-MC3-DMA, DOPG, cholesterol, and PEG-DMG in anhydrous ethanol according to the formula amount, adjust the concentration of the lipid components in the organic phase to 6 mg / mL to obtain an organic phase.

[0113] (c) Mix the aqueous phase of step (a) and the organic phase of step (b) according to a volume ratio of 1:3, using a microfluidic device, at a flow rate of 12 mL / min, immediately dilute the mixture 100 times with a PBS solution at pH 7.4, and remove the ethanol component in the solution using tangential flow filtration (TFF), and then concentrate to a concentration of 55 ug / ml of mRNA in the system to obtain a lipid nanoparticle comprising RNA encoding a respiratory syncytial virus antigen.

[0114] Example 2

[0115] Using luciferase as a reporter gene, the efficiency of different vaccine carrier formulations (as shown in Table 1 below, "MC3" refers to Dlin-MC3-DMA, "+" indicates that luciferase expression in mice was detected by a small animal live fluorescence imaging system after administration) in delivering luciferase gene-encoding mRNA in mice was studied by live fluorescence imaging technology, and the physicochemical indicators of different complex formulations (preparation method, see Example 1) were detected, and the results are shown in Table 1.

[0116] Table 1

[0117]

[0118] It was found through research that increasing the mass ratio of lipid to mRNA is beneficial to increasing the encapsulation rate of mRNA in the lipid nanoparticle, thereby making it have higher stability, and in addition, moderately increasing the content of polyethylene glycol (PEG) in the formulation is beneficial to increasing the expression efficiency of mRNA in vivo. Therefore, considering factors such as mRNA encapsulation rate and mRNA in vivo delivery efficiency, Formulations No. 3 and No. 4 were selected for subsequent mRNA vaccine research.

[0119] Example 3

[0120] The ability of cationic lipid nanoparticles of different formulations to encapsulate luciferase-encoding mRNA and the particle size data of the nanoparticles formed are shown in Table 2. Several formulations can compress luciferase mRNA into nanoparticles with a particle size of less than 100 nm and a net surface potential of zero, and can encapsulate at least 50% of mRNA, thus having certain in vivo delivery effect. "MC3" refers to Dlin-MC3-DMA.

[0121] Table 2

[0122]

[0123] Example 4

[0124] A series of mRNA sequence information was designed as shown in the table. In addition to the open reading frame coding region described in the table, the mRNA sequence characteristics of samples 1-6 also include 5' UTR as shown in SEQ ID No. 11, 3' UTR as shown in SEQ ID No. 14, and 100 polyA, 5' cap is m7Gppp(5')(2'-OMeA)pG, and uracil in the mRNA is replaced with 5' pseudouracil.

[0125] The detailed method is as follows: the HEK293 cells transfected with each mRNA for 24 hours were lysed, 10 μg of total protein was loaded on the SDS-PAGE gel, immunoblotting was performed with anti-SARS-S1 protein antibody, and then labeled with goat anti-mouse-HRP secondary antibody, followed by color development. The protein expression amount was quantified using the internal reference b-actin, and the differences in protein expression amount after transfection of different mRNAs were compared. Cells not transfected with mRNA were used as negative control. The expression results of samples 1-6 are shown in the table.

[0126] As can be seen from the results shown in the table, the S protein expression in the cells transfected with the six different sequence designed mRNAs was expressed, and the full-length band of S protein can be seen according to the molecular weight.

[0127] Table 3

[0128] Sample No. ORF nucleotide sequence Relative expression amount 1 Seq ID NO. 2 1.1 2 Seq ID NO. 3 1.1 3 Seq ID NO. 4 0.6 4 Seq ID NO. 5 0.5 5 Seq ID NO. 6 1.0 6 Seq ID NO. 7 1.0

[0129] Sample 1 and 2 contain mRNA encoding S protein of coronavirus XBB.1.5 variant, which has K982P substitution and V983P substitution compared with wild type S protein of coronavirus XBB.1.5 variant; sample 3 and 4 contain mRNA encoding S protein of coronavirus XBB.1.5 variant, which has F813P substitution, A888P substitution, A895P substitution, A938P substitution, K982P substitution and V983P substitution compared with wild type S protein of coronavirus XBB.1.5 variant; sample 5 and 6 contain mRNA encoding wild type S protein of coronavirus XBB.1.5 variant.

[0130] The expression amount of sample 3 and 4 is lower than that of sample 1-2 and sample 5-6, and the S protein of coronavirus XBB.1.5 variant encoded by sample 3 and 4 has mutations that form mRNA which is not suitable as a potential vaccine selection.

[0131] After transfecting cells with sample 1-2, the S protein content under the same conditions is slightly lower than that of sample 5-6, indicating that K982P substitution and V983P substitution do not significantly improve the expression amount of S protein in the transfection experiment.

[0132] Based on the above results, other sites of SEQ ID No. 1 were also mutated in combination with other published reports, such as G601S, and the open reading frame of the corresponding mRNA sequence was designed based on the open reading frame of the mRNA sequence of sample 1-2, and the protein expression immunoblotting results after mRNA transfection of cells were detected, and it was found that these mutations and the optimization of the corresponding mRNA sequence had lower expression than the mRNA sequence of sample 1-2 and the mRNA sequence of sample 5-6.

[0133] Based on the full-length S protein mRNA of SARS-COV-2 disclosed in WOUS21032609, GB2002166, WOUS21016979, etc., and the optimization method of sample 1-2, the protein expression immunoblotting results after mRNA transfection of cells were detected, and it was found that these mutations and the optimization of the corresponding mRNA sequence had lower expression than the mRNA sequence of sample 1-2 and the mRNA sequence of sample 5-6.

[0134] Example 5

[0135] The samples 1-2 and 5-6 described in Example 4 were respectively formulated into vaccines with the delivery formulation, and different groups of samples were respectively prepared into LNP formulations. The samples 7-11 were respectively formulated into vaccines with the delivery formulation, and different groups of samples were respectively prepared into LNP formulations. Among them, sample 7 encodes the S protein of the original strain of the new coronavirus, and the RNA open reading frame nucleotide sequence contained therein is as shown in Seq ID NO. 17. Samples 8-11 encode the S protein of the new coronavirus Omicron XBB.1.5 with the amino acid sequence as shown in Seq ID NO. 1, wherein sample 8 contains the RNA open reading frame nucleotide sequence as shown in Seq ID NO. 8; sample 9 contains the RNA open reading frame nucleotide sequence as shown in Seq ID NO. 9; sample 10 contains the RNA open reading frame nucleotide sequence as shown in Seq ID NO. 10; and sample 11 contains the RNA open reading frame nucleotide sequence as shown in Seq ID NO. 18.

[0136] In addition to the open reading frame coding region, the mRNA sequence characteristics of samples 1-2 and 5-11 also include a 5' UTR as shown in SEQ ID No. 11, a 3' UTR as shown in SEQ ID No. 14, and 100 polyA, a 5' cap of m7Gppp(5')(2'-OMeA)pG, and uracil in the mRNA is replaced with 5' pseudouracil.

[0137] The nine groups of vaccine formulations obtained were used for C57 mouse immunization experiments, and each mouse was injected with 5 micrograms (in terms of mRNA) through the lateral thigh muscle of the hind leg, and the second immunization was performed after 7 days. The mouse serum was extracted 14 days after the first immunization and sent to a third-party laboratory for SARS-CoV-2 pseudovirus neutralization activity testing. The mouse serum was diluted at different ratios (the initial dilution ratio was 30) in a 96-well plate, and the corresponding infectable SARS-CoV-2 pseudovirus was added, and at the same time, cell controls and virus controls were set up, incubated for 1 hour, and then the prepared cells were added. Incubate in a cell incubator for 20-28 hours, discard part of the supernatant, add luciferase detection reagent, react at room temperature in the dark, repeatedly blow and suck the cells in the hole to make the cells fully lysed, and then place them in a chemiluminescence detector to read the luminescence value. Under the premise that the virus control and the cell control are established, the EC50 value is calculated by the Reed-Muench method. The serum corresponding to the group number and the results are shown in Figure 1 and Figure 2 .

[0138] from Figure 1The results of the experiment of the new coronavirus Omicron XBB.1.5 sub-variant pseudovirus showed that the vaccine preparations prepared from the six samples could stimulate mice to produce antibodies with neutralizing ability to the new coronavirus Omicron XBB.1.5 sub-variant pseudovirus. The neutralizing activity of the vaccine preparation prepared from sample 7 was lower; the neutralizing activity of the vaccine preparations prepared from samples 1-2 and sample 8 was significantly higher than that of the vaccine preparation prepared from sample 7 (P<0.05) and the vaccine preparations prepared from samples 5-6 (P<0.05). The neutralizing activity of the vaccine preparation prepared from sample 1 was significantly higher than that of the vaccine preparations prepared from samples 2 and 8 (P<0.05).

[0139] By Figure 2 The results of the experiment of the new coronavirus Omicron XBB.1.16 sub-variant pseudovirus showed that the vaccine preparations prepared from the five samples could stimulate mice to produce antibodies with neutralizing ability to the new coronavirus Omicron XBB.1.16 sub-variant pseudovirus. The neutralizing activity of the vaccine preparation prepared from sample 7 was lower; the neutralizing activity of the vaccine preparations prepared from samples 1 and samples 9-11 was significantly higher than that of the vaccine preparation prepared from sample 7 (P<0.05); the neutralizing activity of the vaccine preparation prepared from sample 1 was significantly higher than that of the vaccine preparations prepared from samples 9-11 (P<0.05).

[0140] Example 6

[0141] The sample 1 described in Example 4 and the sample 7 described in Example 5 were respectively made into vaccines with the delivery preparation.

[0142] In addition to the open reading frame coding region, the mRNA sequence characteristics of sample 1 and sample 7 also include 5' UTR as shown in SEQ ID No. 11, 3' UTR as shown in SEQ ID No. 14 and 100 polyA, 5' cap is m7Gppp(5')(2'-OMeA)pG, and uracil in the mRNA is replaced with 5' pseudouracil.

[0143] The two vaccine formulations were used in C57 mouse immunization experiments. Each mouse received a 5 microgram (mRNA) intramuscular injection in the lateral thigh of the hind limb, followed by a second immunization 7 days later. Fourteen days after the first immunization, mouse serum was collected 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 pseudovirus was added. Cell controls and virus controls were also included. After incubation for 1 hour, pre-prepared cells were added, and the plates were 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. Serum groupings and results are shown below. Figure 3 As shown.

[0144] Depend on Figure 3 The results of the pseudovirus experiments on the three sub-variants of SARS-CoV-2 (Omeprone) showed that the vaccine formulations prepared from both samples could stimulate mice to produce antibodies with neutralizing ability against the three sub-variants of SARS-CoV-2 (Omeprone). However, the vaccine formulation prepared from sample 7 had lower neutralizing activity; the vaccine formulation prepared from sample 1 had significantly higher neutralizing activity than that prepared from sample 7 (P<0.05). Furthermore, because the S protein of the SARS-CoV-2 (Omeprone) XBB1.5 sub-variant is significantly different from that of the SARS-CoV-2 (Omeprone) BA.5 sub-variant, the neutralizing activity of sample 1 in mice against the SARS-CoV-2 (Omeprone) BA.5 sub-variant was significantly lower than that against the SARS-CoV-2 (Omeprone) XBB.1.5 and XBB.1.16 sub-variant pseudoviruses (P<0.05).

[0145] Example 7

[0146] The sample 1 described in Example 4 and the delivery formulation were used to prepare a vaccine. COVID-19 bivalent vaccine sample 12 was prepared according to the method described in Example 7 of Chinese Patent ZL.2022112076839 for sample 1.

[0147] In addition to the open reading frame coding region, the mRNA sequence features of sample 1 also include a 5' UTR as shown in SEQ ID No. 11, a 3' UTR as shown in SEQ ID No. 14, and 100 polyA sequences. The 5' cap is m7Gppp(5')(2'-OMeA)pG, and uracil in the mRNA is replaced with 5' pseudouracil.

[0148] The two groups of vaccine preparations obtained were used for C57 mouse immunization experiments, and each mouse was injected with 5 micrograms (mRNA) through the lateral thigh muscle of the hind leg, and the second immunization was performed after 7 days. 14 days after the first immunization, the mouse serum was extracted and sent to a third-party P3 laboratory for operation according to the technical specifications of the novel coronavirus "micro- serum neutralization test (FRNT method)", and the neutralization activity of the serum to the live viruses of the three sub-variant strains of the novel coronavirus Omicron BA.5, XBB.1.9 and XBB.1.16 was detected. The results are shown in Figure 4 .

[0149] From Figure 4 The experimental results of the three sub-variant strains of the novel coronavirus Omicron live virus show that the vaccine preparations prepared from the two samples can stimulate mice to produce antibodies with neutralizing activity to the three sub-variant strains of the novel coronavirus Omicron live virus. For the novel coronavirus Omicron BA.5 sub-variant strain, the neutralizing activity of the antibodies produced by sample 12 is much higher than that of the antibodies produced by sample 1; for the novel coronavirus Omicron XBB.1.9 sub-variant strain and the novel coronavirus Omicron XBB.1.16 sub-variant strain, the neutralizing activity of the antibodies produced by sample 1 is higher than that of the antibodies produced by sample 12.

[0150] Example 8

[0151] The construct tested in this example is a plasmid with T7 polymerase co-transfected with Norwood's DNA to activate the promoter on Norwood's 2019-NCOV plasmid in trans. The assay conditions are as follows:

[0152] DNA capable of transcribing the mRNA of sample 1 in Example 4 was prepared, and HEK293 cells were transfected with the DNA to detect the expression of S full-length protein in the cells. The detailed method is as follows: the HEK293 cells transfected with each DNA for 24 hours were lysed, and 10 μg of total protein was loaded on the SDS-PAGE gel, and immunoblotting was performed with anti-SARS-S1 protein antibody. The secondary antibody was goat anti-mouse-HRP, and then color development was performed. The protein expression was quantified using the internal reference b-actin, and the protein expression difference after transfection of different mRNA was compared. Cells without DNA transfection were used as negative control. The experimental results are shown in Figure 5 , and the mRNA of sample 1 can normally express the S protein of the novel coronavirus Omicron XBB.1.5 after being transcribed into DNA. Figure 5 ① indicates the intracellular expression of the S protein of the novel coronavirus Omicron XBB.1.5; ② indicates the extracellular expression of the S protein of the novel coronavirus Omicron XBB.1.5; ③ is Maker; ④ is the intracellular expression of the negative control; and ⑤ is the extracellular expression of the negative control.

[0153] The above merely provides the preferred embodiments of the present application, and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the principles and technical scope of the present application shall fall into the scope of the present application.

Claims

1. A vaccine for coronavirus, characterized in that, The novel coronavirus vaccine is a novel coronavirus nucleic acid vaccine; the novel coronavirus nucleic acid vaccine comprises an mRNA molecule encoding a novel coronavirus antigen; the antigen is a novel coronavirus XBB.1.5 variant S protein, and the novel coronavirus XBB.1.5 variant S protein comprises a K982P substitution and a V983P substitution; the amino acid sequence of the novel coronavirus XBB.1.5 variant S protein is shown in Seq ID NO.

1.

2. The novel coronavirus vaccine as claimed in claim 1, wherein, The mRNA molecule comprises an open reading frame, and the nucleotide sequence of the open reading frame is shown in Seq ID NO. 2-10.

3. The novel coronavirus vaccine as claimed in claim 2, wherein, The mRNA further comprises at least one of a 5' end cap, a 5' UTR, a 3' UTR, and a 3' end poly A tail. The 5' end cap is selected from ARCA, mCAP, dmCAP, tmCAP, m7G(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, dmCAP, or m7G(5')ppp(5')(2'OMeG)pG; the 3' end poly A tail has a length of 50-200 nt; the 5' UTR nucleotide sequence is shown in Seq ID NO. 11-13; the 3' UTR sequence is shown in Seq ID NO. 14-16; and one or more uridines in the mRNA are replaced with modified nucleosides.

4. The novel coronavirus vaccine as claimed in claim 1, wherein, The novel coronavirus vaccine comprises a delivery formulation. The delivery formulation comprises a lipid nanoparticle or a cationic liposome.

5. A method of preparing the novel coronavirus vaccine as claimed in claim 1, wherein, The mRNA and the delivery formulation are mixed to form the novel coronavirus vaccine; the delivery formulation comprises a lipid nanoparticle or a cationic liposome.

6. An isolated mRNA, comprising, The open reading frame of the mRNA encodes a novel coronavirus XBB.1.5 variant S protein, and the amino acid sequence of the novel coronavirus XBB.1.5 variant S protein comprises a K982P substitution and a V983P substitution; the amino acid sequence of the novel coronavirus XBB.1.5 variant S protein is shown in Seq ID NO.

1.

7. An isolated DNA, comprising a nucleotide sequence encoding a polypeptide of claim 1. The DNA is obtained by reverse transcription of the isolated mRNA according to claim 6, or the DNA is a sequence capable of transcribing the isolated mRNA according to claim 6.

8. A biomaterial, characterized by, The biological material comprises any one of an expression cassette, a vector, an engineered bacterium, or a cell line, and the biological material contains or expresses the isolated mRNA according to claim 6 or the isolated DNA according to claim 7.

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