CN116768988A

CN116768988A - mRNA vaccine encoding novel coronavirus S protein

Info

Publication number: CN116768988A
Application number: CN202210224804.4A
Authority: CN
Inventors: 威廉·贾; 刘晓虎; 陆昌瑞; 杨晓明; 张云涛; 侯富军; 杨国环; 程新华; 余志斌; 丁隽; 林康; 徐江; 华先武; 潘欣萍; 刘连晓
Original assignee: Zhongsheng Funuojian Biotechnology Shanghai Co ltd
Current assignee: Zhongsheng Funuojian Biotechnology Shanghai Co ltd
Priority date: 2022-03-09
Filing date: 2022-03-09
Publication date: 2023-09-19
Also published as: WO2023169506A1

Abstract

The present application provides mRNA vaccines encoding novel coronavirus S proteins. The S protein encoded by the mRNA comprises an amino acid sequence shown in any one of SEQ ID NO. 7-8. The mRNA comprises the nucleotide sequence shown in any one of SEQ ID NO. 1-3. The three mRNAs provided by the application can be translated in cells to generate high-level novel coronavirus S protein, and each mRNA is injected into a mouse body through a preparation formed by encapsulating liposome nano particles to induce the mouse to generate high-titer neutralizing antibodies.

Description

mRNA vaccine encoding novel coronavirus S protein

Technical Field

The application belongs to the technical field of biological vaccines, and particularly relates to an mRNA vaccine for encoding novel coronavirus S protein and application thereof.

Background

Vaccines are the most effective public health intervention to prevent infectious diseases. World health organization estimates that two hundred to three million people die each year due to vaccination. Although traditional vaccine types have played a tremendous role in the public health industry, humans still face the threat of new infectious diseases and variants thereof. The development of vaccines against novel infectious diseases, the innovation of vaccine production modes, the improvement of the effectiveness of the vaccines and the shortening of the production period of the vaccines are urgent demands for promoting the progress of public health industry.

Coronaviruses are single-stranded positive strand RNA viruses, and the genome is wrapped by an outer envelope structure f, so that the coronaviruses have the characteristics of strong transmission capacity, easy variation and the like, can infect various animals, and can cause severe acute respiratory syndrome in human beings.

At present, a plurality of different vaccine development routes are simultaneously carried out, such as recombinant protein vaccines, inactivated vaccines and vector vaccines. The production of the recombinant protein vaccine requires large-scale in vitro cell culture, and the antigen protein is separated and purified from the culture, so that the process requirements on protein expression and purification are high; the inactivated vaccine needs to screen proper strains, has long time consumption, has a certain risk in culturing live viruses, and has higher technological requirements on inactivating the viruses. Inactivated viruses are also at risk of reversion and Antibody Dependent Enhancement (ADE).

mRNA vaccines have high safety and ductility and are easy to increase the yield, so that the mRNA vaccine is concerned by scientific researchers and medical institutions, and has wide prospects in vaccine development and production.

The S protein (spike) is an important surface protein of novel coronaviruses. The RBD domain on the S protein binds to the ACE2 protein on the surface of human cells, thereby mediating the entry of SARS-CoV-2 virus into the cells. The S protein is the primary site of action for neutralizing antibodies in the host. The pseudo virus neutralization test shows that the neutralizing antibody aiming at the S protein reduces the positive rate of virus infected cells. Thus, the S protein is the most predominant antigen protein for the development of novel coronavirus vaccines. mRNA vaccines, which have been marketed by Moderna and BioNtech, both encode the full length of the S protein and exhibit extremely high protective efficacy and safety in mass vaccination. However, various mutant strains of SARS-CoV-2 have been detected, such as Alpha, beta, gamma, delta, omacron strains. These mutant strains have a stronger infectivity and transmissibility than the wild-type strains. There is an urgent need to develop mRNA vaccines that are effective against SARS-CoV-2 wild strains and various mutant strains.

Disclosure of Invention

The application provides an RNA encoding a novel coronavirus S protein, and a vaccine comprising the RNA. The mRNA or vaccine of the application has one or more of the following characteristics: (1) higher protein expression, (2) being able to stimulate a stronger immune response, (3) producing more cytokines, (4) having reduced immunogenicity, (5) producing higher levels of neutralizing antibodies, and/or (6) producing immune responses that are protective against both wild-type strains and various mutants of the novel coronavirus.

In one aspect, the application provides an RNA encoding a novel coronavirus S protein, wherein the novel coronavirus S protein comprises the amino acid sequence shown in any one of SEQ ID NO. 7-8. In certain embodiments, the amino acid sequence of the novel coronavirus S protein is set forth in any one of SEQ ID NOS.7-8. In certain embodiments, the S protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID nos. 7-8.

In certain embodiments, the RNA is mRNA.

In certain embodiments, the nucleotide sequence of the RNA is codon optimized.

In certain embodiments, the RNA comprises a nucleotide sequence set forth in any one of SEQ ID nos. 13-15. In certain embodiments, the nucleotide sequence of the RNA is set forth in any one of SEQ ID NO. 13-15. In certain embodiments, the sequence of the RNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the nucleotide sequence set forth in any one of SEQ ID nos. 13-15.

In certain embodiments, the RNA comprises one or more structures selected from the group consisting of: a 5' cap structure, a 5' untranslated sequence, an open reading frame, a 3' untranslated sequence, and a poly (a) tail.

In certain embodiments, the poly (a) tail of the RNA comprises more than 30 adenylates.

In certain embodiments, the RNA has, from the 5 'end to the 3' end, a poly (a) tail having the structure: 30 adenylates, a linker sequence and 70 adenylates. For example, the poly (A) tail can be 100-200 nucleotides in length, such as about 120 nucleotides. Such as about 110 nucleotides.

In certain embodiments, the linking sequence may be 5-20 nucleotides in length. In certain embodiments, the linker sequence is GCAUAUGACU.

In certain embodiments, the poly (A) tail of the RNA comprises the nucleotide sequence set forth in SEQ ID NO. 12. In certain embodiments, the poly (a) tail has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID No. 12.

In certain embodiments, the RNA comprises a nucleotide sequence as set forth in any one of SEQ ID nos. 1-3. In certain embodiments, the sequence of the RNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the nucleotide sequence set forth in any one of SEQ ID nos. 1-3.

In certain embodiments, the RNA comprises at least one modified nucleotide.

In certain embodiments, the RNA comprises a modification at one or more positions selected from the group consisting of: a 5' cap structure, a 5' untranslated sequence, an open reading frame, a 3' untranslated sequence, and a poly (a) tail. For example, the 5 'untranslated sequence may comprise a 5' untranslated sequence of a gene selected from the group consisting of: beta-globin gene, heat shock protein 70 gene, axin heavy chain 2 gene, hydroxysteroid (17-beta) dehydrogenase gene, and/or KOZAK sequence. For example, the 3 'untranslated sequence may comprise a 3' untranslated sequence of a gene selected from the group consisting of: albumin gene, alpha-globin gene, beta-globin gene, tyrosine hydroxylase gene, heat shock protein 70 gene, lipoxygenase gene and collagen alpha gene.

In certain embodiments, the chemical structural formula of the first and second building blocks in the 5' cap structure is as follows:

in certain embodiments, the modified nucleotides of the RNA comprise one or more nucleotides comprising a nucleotide selected from the group consisting of: N1-Methyl pseudouridine triphosphate (N1-Methyl pseudouridine triphosphate-UTP), pseudouridine triphosphate (pseudouridine-UTP), 5-Methoxy uridine triphosphate (5-Methoxy-UDP) and 5-Methyl cytidine triphosphate (5-Methyl-CTP).

In certain embodiments, the structural U bases in the sequence of the RNA are all replaced with m1ψ=1-methyl-3' -pseudoundyhl.

In another aspect, the present application provides a DNA encoding a novel coronavirus S protein, wherein the novel coronavirus S protein comprises the amino acid sequence shown in any one of SEQ ID NOS.7-8

In another aspect, the application provides a DNA for transcribing RNA of the application.

In certain embodiments, the DNA is codon optimized. In certain embodiments, the DNA comprises the nucleotide sequence set forth in any one of SEQ ID nos. 9-11. In certain embodiments, the nucleotide sequence of the DNA is set forth in any one of SEQ ID NOS.9-11. In certain embodiments, the sequence of the DNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the nucleotide sequence set forth in any one of SEQ ID nos. 9-11.

In certain embodiments, the DNA further comprises a 5 'untranslated sequence, a 3' untranslated sequence, and a poly (a) tail.

In certain embodiments, the DNA comprises the nucleotide sequence set forth in any one of SEQ ID nos. 4-6. In certain embodiments, the nucleotide sequence of the DNA is set forth in any one of SEQ ID NOS.4-6. In certain embodiments, the sequence of the DNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the nucleotide sequence set forth in any one of SEQ ID nos. 4-6.

The RNAs (or DNAs) of the application may be formulated in nanoparticles or other delivery vehicles, for example, to avoid their degradation upon delivery to a subject. In the present application, the mRNA may be encapsulated within a nanoparticle. In certain embodiments, the nanoparticle comprises a lipid. Lipid nanoparticles may include, but are not limited to, liposomes and micelles. In the present application, the lipid nanoparticle may comprise cationic and/or ionizable lipids, anionic lipids, neutral lipids, amphiphilic lipids, pegylated lipids, and/or structural lipids, or a combination thereof, in certain embodiments, the lipid nanoparticle comprises one or more RNAs, such as mRNA, as well as mRNA encoding an S protein, as described herein.

With respect to the specific manner of delivery, there is no particular limitation, and those conventionally used in the art may be employed. For example, reference may be made to the manner disclosed in published patent US20160376224A1 or WO2015199952 A1.

The delivery vehicle in the compositions of the present application may be a nanolipid particle. The nanolipid particles may comprise one or more (e.g., 1,2, 3, 4, 5, 6, 7, or 8) cationic and/or ionizable lipids. "cationic lipid" generally refers to a lipid that carries any number of net positive charges at a certain pH (e.g., physiological pH). The cationic lipids may include, but are not limited to, SM102, 3- (didodecylamino) -N1, N1, 4-triacontanyl-1-piperazineethylamine (KL 10), N1- [2- (behenyl amino) ethyl ] -N1, N4, N4-triacontanyl-1, 4-piperazineethylamine (KL 22), 14, 25-tricosyl-15,18,21,24-tetraazaocta-nane (KL 25), DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, octyl-CLinDMA (2S), DODAC, DOTMA, DDAB, DOTAP, DOTAP.C1, DC-Choi, DOSPA, DOGS, DODAP, DODMA, and DMRIE.

In certain embodiments, the molar ratio of the cationic lipid in the lipid nanoparticle is about 40-70%, e.g., about 40-65%, about 40-60%, about 45-55%, or about 48-53%.

In the present application, the nanolipid particles may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) non-cationic lipids. The non-cationic lipid may comprise an anionic lipid. Anionic lipids suitable for use in the lipid nanoparticle of the present application may include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphospholipid ethanolamine, N-succinylphospholipid ethanolamine, N-glutaryl phosphatidyl phosphoethanoi, and other neutral lipids having anionic groups attached thereto.

The non-cationic lipid may comprise a neutral lipid. Neutral lipids suitable for use in the lipid nanoparticle of the present application may include phospholipids, such as distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl base oil phosphatidylcholine (POPC), palmitoyl base oil acyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof. In addition, lipids having a mixture of saturated and unsaturated fatty acid chains may be used. For example, the neutral lipids described herein may be selected from DOPE, DSPC, DPPC, POPC or any related phosphatidylcholine.

In certain embodiments, the molar ratio of the phospholipid in the lipid nanoparticle is about 5-20%.

In the present application, the nano-lipid particles may comprise lipid conjugates, for example, polyethylene glycol (PEG) modified lipids and derivatized lipids. PEG modified lipids can include, but are not limited to, polyethylene glycol chains of up to 5kDa in length covalently attached to lipids having alkyl chains of C6-C20 length. The addition of these components can prevent lipid aggregation, can also increase circulation duration, facilitate delivery of the lipid-nucleic acid composition to target cells, or rapid release of nucleic acid. For example, the polyethylene glycol (PEG) modified lipid molecule may be a PEG-ceramide with a shorter acyl chain (e.g., C14 or C18). In certain embodiments, the molar ratio of the polyethylene glycol (PEG) modified lipid molecules in the lipid nanoparticle is about 0.5-2%, e.g., about 1-2%, about 1.2-1.8%, or about 1.4-1.6%. In certain embodiments, the polyethylene glycol (PEG) modified lipid molecule may be PEG2000-DMG.

In the present application, the nano-lipid particles may further comprise cholesterol. In certain embodiments, the cholesterol is present in the lipid nanoparticle in a molar ratio of about 30-50%, for example, about 35-45%, or about 38-42%.

In the present application, the nano-lipid particles may include cationic lipids, cholesterol, phospholipids, and polyethylene glycol modified lipid molecules. In certain embodiments, the molar ratio of cationic lipid, cholesterol, phospholipid, and polyethylene glycol modified lipid molecule may be 45 to 55: 35-45: 5-15: 0.5 to 2.

In another aspect, the application provides a composition comprising the RNA, and a delivery vehicle.

In another aspect, the application provides a composition that may comprise an mRNA of the application, and may further comprise a delivery vehicle.

In certain embodiments, the delivery vehicle comprises a liposome.

In certain embodiments, the delivery vehicle comprises a Lipid Nanoparticle (LNP).

In certain embodiments, the lipid nanoparticle comprises a cationic lipid, a non-cationic lipid, and cholesterol.

In another aspect, the application provides a lipid nanoparticle formulation that encapsulates the RNA.

In another aspect, the application provides a vaccine comprising said RNA, said DNA, said composition, and/or said lipid nanoparticle formulation. In certain embodiments, the vaccine comprises an RNA vaccine, a DNA vaccine, a recombinant vaccine, and/or an adenovirus vaccine.

In another aspect, the application provides a method of preparing an RNA vaccine, the method comprising: a. dissolving ionized lipid, pegylated lipid, cholesterol and derivatives and phospholipids in ethanol solution; b. mixing the lipid ethanol solution with an RNA aqueous solution by a microfluidic mixer to obtain Lipid Nanoparticles (LNP); c. isolating and purifying the LNP obtained in step b to obtain said RNA vaccine, wherein said RNA is as defined in the present application.

In another aspect, the application provides a pharmaceutical composition comprising said RNA, said DNA, said composition, said liposomal nanoparticle formulation, said vaccine, and/or a pharmaceutically acceptable carrier thereof.

In another aspect, the present application provides a method of treating and/or preventing a disease or disorder associated with a novel coronavirus infection, the method comprising administering the RNA, the DNA, the composition, the liposomal nanoparticle formulation, the vaccine, and/or the pharmaceutical composition to a patient in need thereof.

In some embodiments, the pharmaceutical compositions of the application may be administered to a subject by any method known to those of skill in the art, such as parenteral, oral, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intraventricular, intracranial, intravaginal, or intratumoral.

In the present application, the RNA, the DNA, the vector, the cell, the composition, the liposome nanoparticle preparation, the pharmaceutical composition, and/or the vaccine may be administered singly, multiply, or consecutively, and all have safety. Re-administration of the composition and/or pharmaceutical composition after the first administration may reduce the onset time. In the present application, the RNA, the DNA, the vector, the cell, the composition, the liposome nanoparticle preparation, the pharmaceutical composition and/or the vaccine may be co-administered with other active substances or therapeutic/prophylactic ingredients. In the present application, the RNA, the DNA, the vector, the cell, the composition, the liposome nanoparticle preparation, the pharmaceutical composition and/or the vaccine may be administered before or after other active substances or therapeutic/prophylactic ingredients.

In a further aspect, the application provides the use of said RNA, said DNA, said composition, said liposomal nanoparticle formulation, said vaccine, and/or said pharmaceutical composition in the manufacture of a medicament for the treatment and/or prevention of a novel coronavirus infection-related disease or disorder.

In certain embodiments, the novel coronavirus infection-related diseases or disorders include pneumonia, e.g., covd-19.

In certain embodiments, the drugs (e.g., the vaccine) may be used sequentially.

In certain embodiments, the drug (e.g., the vaccine) may be administered repeatedly a plurality of times, e.g., two, three, four, or more times.

In certain embodiments, the drug (e.g., the vaccine) may be vaccinated sequentially with other vaccines against the novel coronavirus. Other vaccines against the novel coronavirus may be any other vaccine known in the art that prevents the novel coronavirus, including but not limited to an inactivated vaccine, an mRNA vaccine, a DNA vaccine, a recombinant protein vaccine, and/or an adenovirus vaccine. For example, the agent of the present application (e.g., the vaccine) may be administered first, followed by other vaccines against the novel coronavirus, or may be administered first, followed by administration of the agent of the present application (e.g., the vaccine).

In certain embodiments, when the medicament (e.g., the vaccine) is administered sequentially, the time interval between two consecutive administrations can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. The specific time may be determined according to the constitution, health condition, immune response level after administration of the first vaccine, etc. of the subject.

In another aspect, the present application provides a vector comprising said RNA and said DNA.

For example, the vector may be a viral vector, e.g., an adenovirus vector, an adeno-associated virus vector, and/or a lentiviral vector.

In another aspect, the application provides a cell comprising the nucleic acid molecule, and/or the vector. In the present application, the cell may be a prokaryotic cell, for example, E.coli. In the present application, the cells may be eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells. In the present application, the cells may be mammalian cells, such as mouse cells, human cells, and the like.

In certain embodiments, the vector comprises a viral vector.

In another aspect, the application provides a kit comprising said RNA, said DNA, said composition, said liposomal nanoparticle formulation, said vaccine, and/or said pharmaceutical composition.

In another aspect, the application provides a method of producing antibodies against a novel coronavirus comprising administering the RNA, the DNA, the composition, the liposomal nanoparticle formulation, the vaccine, and/or the pharmaceutical composition.

In another aspect, the application provides a method of activating immunity comprising administering the RNA, the DNA, the composition, the liposomal nanoparticle formulation, the vaccine, and/or the pharmaceutical composition.

In certain embodiments, the method is an in vitro or ex vivo method.

Compared with the prior art, the application has the beneficial effects that:

1, three mRNAs provided by the application can be translated in cells to generate high-level novel coronavirus S protein, and each mRNA is injected into a mouse body through a preparation formed by encapsulating liposome nano particles to induce the mouse to generate high-titer neutralizing antibodies. The mRNA of the present application is capable of stimulating a stronger immune response and producing higher levels of neutralizing antibodies than other mrnas or mrnas that are not codon optimized.

2, the three mRNAs provided by the application are CVG031, CVG032 and CVG033. All three mRNAs comprise open reading frames for encoding the whole length of the novel coronavirus S protein, the open reading frames of the three mRNAs are subjected to codon optimization, the CVG031 whole length sequence is shown as SEQ ID NO.1, the CVG032 whole length sequence is shown as SEQ ID NO.2, and the CVG033 whole length sequence is shown as SEQ ID NO. 3; CVG031 and CVG033 code for the same protein sequence, and different codon optimization strategies are adopted, and the coded protein sequence is shown as SEQ ID NO. 7; the protein sequence coded by CVG032 is shown as SEQ ID NO. 8. Mutations exist at multiple sites of the Omicron strain S protein. The protein sequences SEQ ID No.7 and SEQ ID No.8 expressed by the mRNA of the present application each contain a plurality of mutations which occur in the S protein of the novel coronavirus Omicron strain, such as A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G4476S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H Y, N6759K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F; the protein sequence SEQ ID NO.8 also contains the R682G, R683S, R685S mutation. In addition, the above SEQ ID NO.7 protein sequence contains an S-2P mutation which can stabilize the S protein conformation in a pre-fusion state, and the stable pre-fusion S protein S-2P mutation has better immunogenicity than the wild-type S protein. The three mRNAs contained a 5' cap structure, a 5' untranslated sequence (5 ' UTR), a 3' untranslated sequence (3 ' UTR), and a poly (A) tail. The U bases of the structural units in the three mRNA sequences were all replaced by m1 ψ (1-methyl-3' -pseudouridyyl).

Drawings

FIG. 1 shows the expression results of mRNA in HEK 293T/17 cells according to example 2 of the present invention.

FIG. 2 shows the result of translation after transfection of HEK 293T/17 cells with mRNA for Western Blot detection in example 2 of the present invention.

FIG. 3 shows particle size and uniformity of mRNA-LNP coated with CVG031 mRNA in example 3 of the present invention at Room Temperature (RT) and after freeze thawing at-80 ℃.

FIG. 4 shows the encapsulation efficiency of mRNA-LNP coated with CVG031 mRNA at Room Temperature (RT) and after freeze-thawing at-80℃in example 3 of the present invention.

FIG. 5 shows the surface potential properties of mRNA-LNP coated with CVG031 mRNA at Room Temperature (RT) and after freeze-thawing at-80℃in example 3 of the present invention.

FIG. 6 shows the neutralizing antibody titer against Omicron pseudovirus in the serum of mice one week after the primary immunization in example 4 of the present invention; the abscissa corresponds to, from left to right, saline, CVG031 3 μg/min, CVG031 10 μg/min, CVG031 30 μg/min.

FIG. 7 shows the neutralizing antibody titer against wild-type strain pseudovirus, delta pseudovirus, omicron pseudovirus in mouse serum after two immunizations for one week in example 4 of the present invention, wherein WT refers to SARS-CoV-2 wild-type strain; the abscissa corresponds to, from left to right, saline, CVG031 3 μg/min, CVG031 10 μg/min, CVG031 30 μg/min.

FIG. 8 shows the neutralizing antibody titers against wild-type strain pseudovirus, delta pseudovirus, omicron pseudovirus in the serum of mice after two weeks of immunization in example 4 of the present application, wherein WT refers to the SARS-CoV-2 wild-type strain; the abscissa corresponds to, from left to right, saline, CVG031 3 μg/min, CVG031 10 μg/min, CVG031 30 μg/min.

FIG. 9 shows that CVG 031-induced neutralizing antibodies cross-react with the Omacron subtype.

FIG. 10 is a diagram showing CVG031 to cause CD4 ⁺ T cells (FIGS. 10A-10D) and CD8 ⁺ Antigen-specific reaction of T (FIGS. 10E-10H) cells, wherein wild-type (FIGS. 10A and 10E), delta (FIGS. 10B and 10F) or Omicon (FIGS. 10C and 10G) Spike protein post-treatment IFN gamma ⁺ /CD69 ⁺ T cell number is increased, delta S protein stimulatedIL2+ lymphocytes were significantly increased in the mouse spleen cells (FIG. 10I), while IL-5 was not elevated (FIG. 10J).

FIG. 11 is protection against SARS-Cov-2 Delta and Omicron variant challenge in CVG031 heterologous boosted mice, where A: delta and Omicron variants challenge the experimental design of protection assays, B and C: survival rate of each group after viral infection; d and E: body weight changes in animals following infection.

Detailed Description

Further advantages and effects of the present application will become readily apparent to those skilled in the art from the present disclosure, by describing embodiments of the present application with specific examples.

In the present application, the term "delivery vehicle" generally refers to a transfer vehicle capable of delivering an agent (e.g., mRNA) to a target cell. The delivery vehicle may deliver the agent (e.g., mRNA) to a particular subset of cells. For example, the delivery vehicle may be targeted to certain types of cells by the inherent characteristics of the delivery vehicle or by a moiety coupled to the vehicle, contained within (or bound to the vehicle such that the moiety and the delivery vehicle remain together such that the moiety is sufficient to target the delivery vehicle). The delivery vehicle may also increase the in vivo half-life of the agent (e.g., mRNA) to be delivered and/or the bioavailability of the agent to be delivered. Delivery vehicles may include viral vectors, virus-like particles, polycationic vectors, peptide vectors, liposomes, and/or hybrid vectors. For example, if the target cell is a hepatocyte, the properties (e.g., size, charge, and/or pH) of the delivery vehicle may be effective to deliver the delivery vehicle and/or molecules (e.g., mRNA) entrapped therein to the target cell, reduce immune clearance, and/or promote residence in the target cell.

In the present application, the term "DNA" generally includes cDNA or genomic DNA, and "RNA" generally includes mRNA, and also includes analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The DNA or RNA may be single-stranded or double-stranded. In the present application, the term "mRNA" generally refers to RNA transcripts that have been treated to remove introns and are capable of being translated into polypeptides.

In the present application, the term "modified" when used with respect to a nucleic acid (e.g., RNA or DNA) generally refers to a nucleic acid having a different nucleotide molecule, a different nucleotide sequence, consisting of different bonds and/or incorporating non-natural moieties into its structure as compared to the corresponding wild type. For example, the modification may include modification of a nucleotide, e.g., the nucleotide may comprise a modified base, sugar, or phosphate group. For example, the modifications may include polypeptides or proteins of different nucleotide sequences but encoding the same amino acid sequence, or polypeptides or proteins of the same function. The modification may be a chemical modification and/or a biological modification. "chemical modification" can include modification that introduces chemicals other than those found in wild-type or naturally occurring nucleic acids, e.g., covalent modification, e.g., introduction of modified nucleotides (e.g., nucleotide analogs, or introduction of side groups not found naturally in such nucleic acid molecules). The term "modified nucleotide" generally refers to a unit in a nucleic acid polymer that contains modified base, sugar, or phosphate groups, or that incorporates non-natural moieties in its structure.

According to the invention, naturally occurring or modified nucleosides and/or nucleotides, or optimized nucleotides, can be used to prepare modified nucleic acids, e.g., modified mRNA. For example, the modified mRNA can include one or more natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); modified nucleosides (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deadenosine, 7-deazaguanosine, 8-oxoguanosine, O (6) -methylguanosine, pseudouridine, (e.g., N-1-methyl-pseudouridine), 2-thiouridine, and 2-thiocytosine); chemically modifying the base; biologically modified bases (e.g., methylated bases); inserting a base; modified sugars (e.g., 2 '-fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose); modified phosphate groups (e.g., phosphorothioate and 5' -N-phosphoramidite linkages), or any combination thereof. These modified nucleotides may be natural nucleotides or may be artificially optimized or modified nucleotides.

An RNA molecule (e.g., mRNA) can include at least 0.1% modified nucleotides. The fraction of modified nucleotides can be calculated as: the number of modified nucleotides/total number of nucleotides x 100%. In some embodiments, the RNA molecule comprises from about 0.1% modified nucleotides to about 100% modified nucleotides. In some embodiments, the RNA molecule comprises about 0.1% modified nucleotide, about 0.2% modified nucleotide, about 0.5% modified nucleotide, about 1% modified nucleotide, about 2% modified nucleotide, about 5% modified nucleotide, about 10% modified nucleotide, about 20% modified nucleotide, about 50% modified nucleotide, or about 100% modified nucleotide.

In the present application, the term "codon optimization" when used with respect to a nucleic acid generally means that the nucleic acid encoding the polypeptide has been modified to have improved expression in the cell, e.g., a mammalian cell or bacterial cell, by replacing one, at least one, or more codons in the parent polypeptide encoding nucleic acid with codons encoding the same amino acid residues that have different relative frequencies of use in the cell.

In the present application, the term "pharmaceutical composition" generally refers to a formulation in a form that is effective to allow the biological activity of the active ingredient (e.g., vaccine, composition, pharmaceutical composition, vector, cell, DNA or RNA of the present application) and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is to be administered. These formulations may be sterile.

In the present application, the term "S protein", which may also be referred to as "Spike protein" or "Spike protein", generally refers to a membrane protein of a coronavirus surface, which may form protruding homotrimers on the virus surface.

In the present application, the term "vaccine" generally refers to an agent or composition containing an active component effective to induce a therapeutic degree of immunity in a subject against a particular pathogen or disease.

In the present application, the term "lipid nanoparticle" generally refers to a particle comprising a plurality of (i.e. more than one) lipid molecules that are physically bound to each other (e.g. covalently or non-covalently) by intermolecular forces. The lipid nanoparticle may be, for example, a microsphere (including unilamellar and multilamellar vesicles, such as liposomes), a dispersed phase in an emulsion, a micelle, or an internal phase in suspension. The lipid nanoparticle may comprise one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids).

In the present application, the term "liposome" generally refers to a vesicle having an interior space that is isolated from an external medium by one or more bilayer membranes. For example, the membrane of the bilayer may be formed by an amphiphilic molecule, such as a lipid of synthetic or natural origin comprising spatially separated hydrophilic and hydrophobic domains; for another example, the membrane of the bilayer may be formed by an amphiphilic polymer and a surfactant.

In the present application, the term "novel coronavirus" generally refers to severe acute respiratory syndrome coronavirus type 2, which is designated as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) throughout the English language. SARS-CoV-2 belongs to the Coronaviridae (Coronaviridae) genus B coronavirus (Betacorovirus) Sha Bei subgenera (Sarbecovirus). SARS-CoV-2 is a enveloped, non-segmented, positive-stranded single-stranded RNA virus. It can cause novel coronavirus pneumonia (COVID-19).

In the present application, the term "pharmaceutically acceptable carrier" generally refers to any adjuvant, excipient or other pharmaceutical carrier that is compatible with the active ingredient being administered, such as solvents, dispersion media, coatings, isotonic and absorption delaying agents.

In the present application, the term "carrier" is intended to include any solvent, dispersion medium, coating, diluent, buffer, isotonic agent, solution, suspension, colloid, inert, and the like, or combinations thereof, that is pharmaceutically acceptable for administration to the relevant animal, or if applicable, for therapeutic or diagnostic purposes.

In the present application, the term "effective amount" refers to an amount capable of treating or ameliorating a disease or condition or capable of producing a desired therapeutic effect.

As used herein, the term "homology" or "identity" refers to the degree of complementarity between two or more polynucleotide or polypeptide sequences. The term "identity" may be substituted for the term "homology" when a first nucleic acid or amino acid sequence has a primary sequence that is identical to a second nucleic acid or amino acid sequence. Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods can be used to assess whether a given sequence has identity or homology to another selected sequence.

In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "percent identity" refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, using one of the sequence comparison algorithms described below (or other algorithms available to the ordinarily skilled artisan), or as measured by visual inspection.

In this context, when it is stated that "SEQ ID NO" means a DNA sequence or an RNA sequence, it also includes another DNA sequence or DNA sequence complementary to the sequence.

In this context, "SEQ ID NOS.1-15" has the meanings given in the following Table:

sequence number	Type(s)	Sequence name	Sequence number
				1	mRNA full length	CVG031 mRNA full length	SEQ ID NO.1
2	mRNA full length	CVG032 full-length mRNA	SEQ ID NO.2
				3	mRNA full length	CVG033 mRNA full length	SEQ ID NO.3
4	DNA full length	DNA full length of CVG031	SEQ ID NO.4
				5	DNA full length	Full length of CVG032 DNA	SEQ ID NO.5
6	DNA full length	Full length of CVG033 DNA	SEQ ID NO.6
				7	S protein	Protein of CVG031 and protein of CVG033	SEQ ID NO.7
8	S protein	Protein of CVG032	SEQ ID NO.8
				9	DNA of CDS	DNA of CVG031 protein coding region	SEQ ID NO.9
10	DNA of CDS	DNA of CVG032 protein coding region	SEQ ID NO.10
				11	DNA of CDS	DNA of CVG033 protein coding region	SEQ ID NO.11
12	poly(A)	RNA-form sequences of poly (A)	SEQ ID NO.12
				13	RNA of CDS	CVG031 mRNA CDS	SEQ ID NO.13
14	RNA of CDS	CVG032 mRNA CDS	SEQ ID NO.14
				15	RNA of CDS	CVG033 mRNA CDS	SEQ ID NO.15

As used herein, the term "kit" may be used to describe a variant of a portable self-contained housing that includes at least one set of the agents, components or pharmaceutically formulated compositions of the present invention. Optionally, such a kit may include one or more sets of instructions regarding the use of the packaged composition, for example, in a laboratory or clinical application.

As used herein, the term "preventing" or "treating" refers to administering a compound alone or in a pharmaceutical composition prior to the onset of clinical symptoms of a disease state to prevent any symptoms, aspects or features of the disease state. Such prevention and inhibition need not be absolutely considered medically useful.

The application also provides one or more of the following embodiments:

1. an RNA encoding a novel coronavirus S protein, wherein the novel coronavirus S protein comprises an amino acid sequence as set forth in any one of SEQ ID nos. 7-8.

2. The RNA of embodiment 1, which is mRNA.

3. The RNA of any one of embodiments 1-2, wherein the nucleotide sequence is codon optimized.

4. The RNA of any one of embodiments 1-3 comprising a nucleotide sequence as set forth in any one of SEQ ID NOS.13-15.

5. The RNA of any one of embodiments 1-4, comprising one or more structures selected from the group consisting of: a 5' cap structure, a 5' untranslated sequence, an open reading frame, a 3' untranslated sequence, and a poly (a) tail.

6. The RNA of embodiment 5, wherein the poly (a) tail comprises more than 30 adenylates.

7. The RNA of embodiment 5 or 6, wherein, from the 5 'end to the 3' end, the poly (a) tail has the structure: 30 adenylates, a linker sequence and 70 adenylates.

8. The RNA of any one of embodiments 5-7, wherein the poly (a) tail comprises the nucleotide sequence set forth in SEQ ID No. 12.

9. The RNA according to any one of embodiments 1-8 comprising a nucleotide sequence as set forth in any one of SEQ ID NOS.1-3.

10. The RNA of any one of embodiments 1-9, comprising at least one modified nucleotide.

11. The RNA of any one of embodiments 1-10, comprising a modification at one or more positions selected from the group consisting of: a 5' cap structure, a 5' untranslated sequence, an open reading frame, a 3' untranslated sequence, and a poly (a) tail.

12. The RNA of embodiment 10 or 11, wherein the modified nucleotide comprises one or more nucleotides comprising a nucleotide selected from the group consisting of: N1-Methyl pseudouridine triphosphate (N1-Methyl pseudouridine triphosphate-UTP), pseudouridine triphosphate (pseudouridine-UTP), 5-Methoxy uridine triphosphate (5-Methoxy-UDP) and 5-Methyl cytidine triphosphate (5-Methyl-CTP).

13. DNA for transcription of the RNA of any one of embodiments 1 to 12, said DNA comprising the nucleotide sequence set forth in any one of SEQ ID NO.9 to 11.

14. The DNA of embodiment 13, wherein the DNA further comprises a 5 'untranslated sequence, a 3' untranslated sequence, and a poly (a) tail.

15. The DNA according to any one of embodiments 13 to 14, which comprises the nucleotide sequence shown in any one of SEQ ID NOS.4 to 6.

16. A composition comprising the RNA of any one of embodiments 1-12, and a delivery vehicle.

17. The composition of embodiment 16, wherein the delivery vehicle comprises a liposome.

18. The composition of any one of embodiments 16-17, wherein the delivery vehicle comprises a Lipid Nanoparticle (LNP).

19. The composition of any of embodiments 16-18, wherein the lipid nanoparticle comprises a cationic lipid, a non-cationic lipid, and cholesterol.

20. A liposomal nanoparticle formulation that encapsulates the RNA of any one of embodiments 1-12.

21. A vaccine comprising the RNA of any one of embodiments 1-12, the DNA of any one of embodiments 13-15, the composition of any one of embodiments 16-19, and/or the lipid nanoparticle formulation of embodiment 20.

22. A pharmaceutical composition comprising the RNA of any one of embodiments 1-12, the DNA of any one of embodiments 13-15, the composition of any one of embodiments 16-19, the liposomal nanoparticle formulation of embodiment 20, the vaccine of embodiment 21, and/or a pharmaceutically acceptable carrier thereof.

23. A method of treating and/or preventing a novel coronavirus infection-related disease or disorder, the method comprising administering the RNA of any one of embodiments 1-12, the DNA of any one of embodiments 13-15, the composition of any one of embodiments 16-19, the liposomal nanoparticle formulation of embodiment 20, the vaccine of embodiment 21, and/or the pharmaceutical composition of embodiment 22 to a patient in need thereof.

24. Use of the RNA of any of embodiments 1-12, the DNA of any of embodiments 13-15, the composition of any of embodiments 16-19, the liposomal nanoparticle formulation of embodiment 20, the vaccine of embodiment 21, and/or the pharmaceutical composition of embodiment 22 in the manufacture of a medicament for the treatment and/or prevention of a novel coronavirus infection-related disease or disorder.

25. A vector comprising the RNA of any one of embodiments 1-12, the DNA of any one of embodiments 13-15.

26. The vector of embodiment 25, comprising a viral vector.

27. A cell comprising the RNA of any one of embodiments 1-12, the DNA of any one of embodiments 13-15, and/or the vector of any one of embodiments 25-26.

28. A kit comprising the RNA of any of embodiments 1-12, the DNA of any of embodiments 13-15, the composition of any of embodiments 16-19, the liposomal nanoparticle formulation of embodiment 20, the vaccine of embodiment 21, and/or the pharmaceutical composition of embodiment 22.

29. A method of producing an antibody against a novel coronavirus comprising administering the RNA of any one of embodiments 1-12, the DNA of any one of embodiments 13-15, the composition of any one of embodiments 16-19, the liposomal nanoparticle formulation of embodiment 20, the vaccine of embodiment 21, and/or the pharmaceutical composition of embodiment 22.

30. A method of activating immunity comprising administering the RNA of any one of embodiments 1-12, the DNA of any one of embodiments 13-15, the composition of any one of embodiments 16-19, the liposomal nanoparticle formulation of embodiment 20, the vaccine of embodiment 21, and/or the pharmaceutical composition of embodiment 22.

31. The method of any one of embodiments 29-30, which is an in vitro or ex vivo method.

Examples

The following describes the technical scheme of the present invention in detail by referring to examples. The reagents and biological materials used hereinafter are commercial products unless otherwise specified.

The specific experimental process of the invention is as follows: 1. obtaining the gene sequence of SARS-CoV-2 virus; 2. obtaining a protein sequence of SARS-CoV-2 virus; 3. designing a DNA template sequence for encoding the antigen protein; 4. in vitro transcription to produce mRNA; 5. the liposome nanoparticle encapsulates the mRNA; 6. immunizing a mouse with a liposomal nanoparticle formulation encapsulating mRNA; 7. neutralizing antibodies to mouse serum were detected.

Example 1 preparation of mRNA

After sequence optimization is carried out on the RNA, a DNA template transcribed in vitro by the RNA is cloned into a pUC57 vector by using an experimental method known by a person skilled in the art, and the RNA is transformed into competent cells of escherichia coli, so that positive cloning strains are preserved. Amplifying the cultured strain, extracting and purifying DNA template plasmid, linearizing the plasmid after enzyme digestion, and carrying out RNA In Vitro Transcription (IVT) by taking the linearized plasmid as a template to purify an RNA product. The method comprises the following specific steps:

The company is entrusted to synthesize three gene sequences encoding the total length of the novel coronavirus S protein, the gene sequences are subjected to codon optimization, the sequences are shown as SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11, the translated protein sequences of the SEQ ID NO.9 and the SEQ ID NO.10 are identical, the sequence is shown as SEQ ID NO.7, and the translated protein sequence of the SEQ ID NO.11 is shown as SEQ ID NO. 8. Preferably, the three genes are synthesized together with a 5'UTR DNA sequence, a 3' UTR DNA sequence, and a poly (A) tail. The synthetic gene products comprising the 5'UTR, open reading frame, 3' UTR, poly (A) tail (sequences shown in SEQ ID NOS.4-6, respectively) were cloned into pUC57 vectors. After amplifying the vector, restriction endonuclease is used for enzyme digestion to obtain a linearization vector. The purified linearized vector was used as a template for RNA in vitro transcription, and T7RNA polymerase, CTP, GTP, ATP, m 1.sup.1ψ (1-methyl-3' -pseudouridyyl) modified UTP, cap analogue, and other necessary components known to those skilled in the art were added thereto, and incubated at 37℃for 1 to 5 hours. After the reaction, the DNA was removed by digestion with DNase and further purified to obtain mRNA product. mRNA concentration and integrity are measured by methods well known to those skilled in the art. Three mRNAs were prepared and named CVG031, CVG032 and CVG033, and the nucleic acid sequences were shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

Example 2 mRNA cell translatability assay

mRNA was prepared according to the procedure described in example 1, and CVG031 mRNA was mixed with the transfection reagent in a directed ratio, and transfected into HEK 293T/17 cells, and the expression of the mRNA after transfection of the cells was detected, as shown in FIG. 1, and the expression of the mRNA in HEK 293T/17 cells was shown in FIG. 1. The full-length expression of the novel coronavirus S protein was detected by Western Blot, and the results of the detected S protein are shown in FIG. 2. Spike in fig. 2 indicates the molecular weight size of the novel coronavirus S protein, ctrl is a negative control group, and no expression of the novel coronavirus S protein was observed; the LNP/CVG031 group, which contains CVG031 mRNA, expresses protein products of the same size as the molecular weight of the new coronavirus S protein. The results of fig. 1 and 2 show that: the CVG031 mRNA can be translated into the S protein of a new coronavirus.

EXAMPLE 3 Liposome nanoparticle coating CVG031 mRNA

The mRNA obtained in example 1 was encapsulated: the cationic lipid (MC 3), DSPC, cholesterol and PEG-lipid were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. Lipid Nanoparticles (LNP) were prepared at a weight ratio of total lipid to mRNA of about 10:1 to 30:1. Briefly, the mRNA and four-component lipid mixture of example 1 was made into mRNA-LNP using a microfluidic nano-precipitation process in which an aqueous mRNA solution at acidic pH was rapidly mixed with an ethanol solution of the lipid. Ethanol was then removed from the crude product by tangential flow ultrafiltration (TFF), followed by buffer exchange using PBS solution (1 x, ph 7.4). Next, mRNA in the neutralized product was diluted to 0.5mg/mL using sucrose solution. Finally, the product was sterile filtered through a 0.22 μm Sartopore PES membrane, and then aliquots were stored at room temperature or frozen at-80 ℃ (1.0 mL fill).

Characterization of mRNA-LNP: the particle size, polydispersity (PDI) and Zeta potential of the mRNA-LNP were measured by dynamic light scattering (Malvern Nano ZS Zetasizer). The diameter is characterized by Z average. The encapsulation efficiency (e.e.) of mRNA in LNP is defined as the mass ratio of mRNA encapsulated to total mRNA in the final mRNA-LNP product. Using specific intercalating fluorescent dyes (Quant-iT ^TM RNA Reagent) to quantitatively detect nucleic acids such as mRNA. mRNA concentrations were calculated from calibration curves generated using mRNA standards. The relative proportion of encapsulated mRNA in mRNA-LNP was determined by the ratio of fluorescent signal in the absence to the presence of the surfactant dispersing LNP. The signal in the absence of surfactant indicates the level of free mRNA, while the signal in the presence of surfactant is used as a measure of total mRNA in the sample.

The particle size and uniformity of the mRNA-LNP coated with CVG031 mRNA at Room Temperature (RT) and after freeze thawing at-80℃are shown in FIG. 3, and the LNP diameters in both cases are almost unchanged and are between 60 and 80nm, indicating that CVG031 mRNA-LNP is very stable. The encapsulation efficiency of mRNA almost reaches 100% after freeze thawing of mRNA-LNP coated with CVG031 mRNA at Room Temperature (RT) and-80 ℃, as shown in FIG. 4, which shows that the novel mRNA crown vaccine preparation prepared by using LNP can encapsulate mRNA in LNP very efficiently and is still very stable after freeze thawing at-80 ℃. After the mRNA-LNP coated with CVG031 mRNA is frozen and thawed at Room Temperature (RT) and at-80 ℃, the surface potential properties of the nano particles are shown as figure 5, and all the nano particles keep negative charge properties, which indicates that the mRNA-LNP can be uniformly suspended and distributed in the solution; the smaller absolute value of charge indicates that mRNA-LNP is still better able to pass through the cell membrane whose outer surface is also negatively charged.

EXAMPLE 4 animal immunization

After the liposome nanoparticle preparation coated with CVG031mRNA of example 3 was filtered, mice were injected, and a control group was injected with physiological Saline (Saline), the injection doses of the liposome nanoparticle preparation were 3. Mu.g/dose, 10. Mu.g/dose, and 30. Mu.g/dose, respectively. Seven days after the first immunization, blood was collected from the mice, and the neutralizing antibody titer against omacron new coronavirus in the serum was measured, and the results are shown in fig. 6. Mice were re-injected on day 21 after primary immunization, which was secondary immunization, using the liposome nanoparticle formulation filtered in example 3. After one week of secondary immunization, the mice were collected and tested for neutralizing antibody titers in serum, and the results are shown in fig. 7. Two weeks after the secondary immunization, the blood of the mice was collected, and the neutralizing antibody titer in the serum was measured, and the results are shown in fig. 8.

The experimental results show that: the CVG031mRNA and liposome nanoparticle prepared by liposome components provided by the application are injected into a mouse body to induce the generation of high-level neutralizing antibodies of the novel omacron mutant coronavirus strain, and the level of the neutralizing antibodies is increased along with the increase of the concentration of the liposome nanoparticle.

The CVG032 mRNA and the CVG033 mRNA with the nucleic acid sequences shown in SEQ ID NO.2 and SEQ ID NO.3 have similar immune effects as the CVG031mRNA, all three mRNAs can be translated in cells to generate high-level novel coronavirus S protein, and each mRNA is injected into a mouse body through a preparation formed by encapsulating liposome nano particles to induce the mouse to generate high-titer neutralizing antibodies. The three mRNAs of the present application are capable of stimulating a stronger immune response and producing higher levels of neutralizing antibodies than other mRNAs or mRNAs that are not codon optimized.

Example 5 CVG 031-induced Cross-reaction of neutralizing antibodies with Omicron subspecies

Mice were immunized twice with CVG031 on day 0 and day 14, and serum samples from animals immunized for the second 14 days were incubated with Omicron ba.1 and ba.2 pseudoviruses and tested for neutralization activity. The results (FIG. 9) show that CVG 031-induced antibodies cross-react with Omicron BA.1 and BA.2.

EXAMPLE 6 CVG031 induces cellular immunity

This example examined T cell immune responses in CVG031 immunized animals. Intracellular staining was performed with cytokines induced by restimulation of the different variant full-length S proteins, and S protein-specific cellular immune function was assessed. Spleen cells were collected 4 weeks after the second inoculation for analysis. The results (FIG. 10) show that CVG031 can cause antigen-specific responses in CD8 and CD4T cells, which can be derived from IFNγ after treatment with Spike proteins of wild type, delta or Omicron, respectively ⁺ /CD69 ⁺ The increase in the number of T cells is seen (fig. 10A-10H). The results indicate that immunization of mice with CVG031 produced a strong immune response to the novel coronavirus. ELSA detection of IL2 expression in spleen cells of S protein stimulated mice also showed a significant increase in IL2+ lymphocytes from CVG 031-vaccinated mice (FIG. 10I). At the same time, IL-5 was not elevated in all samples (FIG. 10J), indicating that CVG031 induced primarily a specific Th1 immune response, but not Th2.

EXAMPLE 7 CVG031 boost

This example tested the protective effect of BBIBP-CorV (Beijing institute of biological products, china biological group) enhanced homologous BBIBP-CorV or heterologous CVG031 enhanced needle vaccination on ACE2 expressing transgenic mice infected with Delta or Omicron mutants. Figure 11A shows that animals were injected with different doses of BBIBP-cov or CVG031 as booster needles 14 days after completion of 2 doses of BBIBP-cov inoculation. These animals were vaccinated with 200 ccid50 doses of Delta or Omicron virus by intranasal absorption 14 days after booster needle injection. In the saline control group, 4/4 animals died within 6 days after infection with the Delta variant. At the same time, 2/4 animals died within 8 days after infection with omacron virus, and the remaining 2 animals developed severe disease (black line, fig. 11B and 11C). Within 8 days after infection of both variants, no animal death occurred in either the vaccinated homologous or heterologous immunization groups. However, in the BBIBP-CorV homologous enhanced needle experimental group, 4/4 animals developed severe symptoms and developed significant weight loss on day 8 after infection with Delta variation (grey line, FIG. 11D).

The foregoing is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications are within the scope of the present invention.

Claims

1. An RNA encoding a novel coronavirus S protein, wherein the novel coronavirus S protein comprises an amino acid sequence as shown in SEQ ID No.7 or SEQ ID No. 8.

2. The RNA of claim 1, comprising a nucleotide sequence as set forth in any one of SEQ ID nos. 13-15.

3. The RNA of any one of claims 1-2, comprising the structure of the group: a 5' cap structure, a 5' untranslated sequence, an open reading frame, a 3' untranslated sequence, and a poly (a) tail.

4. The RNA of claim 3, wherein the poly (a) tail comprises the nucleotide sequence set forth in SEQ ID No. 12.

5. The RNA of any one of claims 1-4, comprising a nucleotide sequence as set forth in any one of SEQ ID nos. 1-3.

6. The RNA of any one of claims 1-5, comprising at least one modified nucleotide.

7. The RNA of claim 6, wherein the modified nucleotide comprises one or more nucleotides comprising a nucleotide selected from the group consisting of: N1-Methyl pseudouridine triphosphate (N1-Methyl pseudouridine triphosphate-UTP), pseudouridine triphosphate (pseudouridine-UTP), 5-Methoxy uridine triphosphate (5-Methoxy-UDP) and 5-Methyl cytidine triphosphate (5-Methyl-CTP).

8. DNA transcribed from the RNA of any of claims 1 to 7 comprising the nucleotide sequence shown in SEQ ID No.9 to 11.

9. The DNA of claim 8 comprising the structure of: a 5 'untranslated sequence, an open reading frame, a 3' untranslated sequence, and a poly (A) tail.

10. The DNA according to any one of claims 8 to 9, comprising the nucleotide sequence set forth in any one of SEQ ID nos. 4 to 6.

11. A liposomal nanoparticle formulation that encapsulates the RNA of any one of claims 1-7.

12. A vaccine comprising the RNA of any one of claims 1-7, the DNA of any one of claims 8-10, and/or the lipid nanoparticle formulation of claim 11.

13. A pharmaceutical composition comprising the RNA of any one of claims 1-7, the DNA of any one of claims 8-10, and/or the lipid nanoparticle formulation of claim 11, the vaccine of claim 12, and/or a pharmaceutically acceptable carrier thereof.

14. Use of the RNA of any one of claims 1-7, the DNA of any one of claims 8-10, and/or the lipid nanoparticle formulation of claim 11, the vaccine of claim 12, and/or the pharmaceutical composition of claim 13 in the manufacture of a medicament for the treatment and/or prevention of a novel coronavirus infection-related disease or disorder.

15. The use of claim 14, the medicament being suitable for sequential administration.