WO2023169500A1 - Mrna vaccine for encoding s protein of sars-cov-2 - Google Patents

Abstract

Provided in the present application is an mRNA vaccine encoding the S protein of SARS-CoV-2. The S protein encoded by the mRNA comprises an amino acid sequence as shown in any one of SEQ ID NOs.5-6. The mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO.1 and SEQ ID NO.2. The two mRNAs provided in the present invention can be translated in cells to generate the high-level S protein of SARS-CoV-2. Each mRNA is injected into the body of a mouse via a preparation formed by means of encapsulating same with a liposome nanoparticle, thereby inducing the mouse to generate a high-titer neutralizing antibody.

Description

mRNA vaccine encoding novel coronavirus S protein Technical field

The invention belongs to the technical field of biological vaccines, and specifically relates to an mRNA vaccine encoding the S protein of the new coronavirus and its application.

Background technique

Vaccines are the most effective public health intervention to prevent infectious diseases. The World Health Organization estimates that between two and three million deaths are averted each year as a result of vaccinations. Although traditional vaccine types have played a huge role in public health, humans still face the threat of new infectious diseases and their mutant strains. Developing vaccines against new infectious diseases, innovating vaccine production methods, improving vaccine effectiveness, and shortening vaccine production cycles are urgent needs to promote the progress of public health.

Coronavirus is a single-stranded positive-sense RNA virus with a genome wrapped by an outer envelope structure. It has the characteristics of strong transmissibility and easy mutation. It can infect a variety of animals and can cause severe acute respiratory syndrome in humans. Entering the 21st century, three highly pathogenic coronaviruses, SARS-CoV, MERS-CoV and SARS-CoV-2, have emerged, posing a serious threat to human health. The global mortality rate of SARS-CoV is 10%, and the global mortality rate of MERS-CoV is 35%. Since its discovery in 2019, the new coronavirus (SARS-CoV-2) has caused a global pandemic. According to the official website of the World Health Organization (WHO), as of January 10, 2022, the number of confirmed cases of COVID-19 worldwide has exceeded 300 million. There were 5.48 million deaths among them. The development of a vaccine against the novel coronavirus is an urgent need to contain the novel coronavirus pandemic.

Currently, multiple different vaccine development routes are underway simultaneously, such as recombinant protein vaccines, inactivated vaccines, and vector vaccines. The production of recombinant protein vaccines requires large-scale in vitro cell culture, and the separation and purification of antigen proteins from the cultures, which requires high protein expression and purification processes; inactivated vaccines require screening of suitable strains, which is time-consuming and requires the cultivation of live viruses. There are certain risks, and the process requirements for inactivating viruses are relatively high. Inactivated viruses also have the risk of atavism and antibody-dependent enhancement (ADE).

mRNA vaccines are highly safe and scalable, and can easily increase production. Therefore, they have attracted the attention of scientific researchers and medical institutions. The application of mRNA in vaccine development and production has broad prospects.

S protein (spike) is an important surface protein of the new coronavirus. The RBD domain on the S protein binds to the ACE2 protein on the surface of human cells, thereby mediating the entry of the SARS-CoV-2 virus into cells. The S protein is the main site of action of neutralizing antibodies in the host. Pseudovirus neutralization experiments showed that neutralizing antibodies against the S protein reduced the ability of the virus to infect cells. Positive rate. Therefore, S protein is the most important antigenic protein for the development of new coronavirus vaccines. The mRNA vaccines already on the market from Moderna and BioNtech both encode the full length of the S protein and have shown extremely high protective efficacy and safety in large-scale vaccination campaigns. However, as the infection time in the population increases, multiple mutant strains of SARS-CoV-2 have been detected, such as Alpha strain, Beta strain, Gamma strain, Delta strain, and Omicron strain. Compared with wild-type strains, these mutant strains have stronger infectivity and transmissibility, and existing new coronavirus vaccines have shown varying degrees of reduced protective efficacy. Therefore, the development of mRNA vaccines that are effective against wild strains and multiple mutant strains of SARS-CoV-2 is an urgent need to curb the epidemic.

Contents of the invention

This application provides an RNA encoding the S protein of the new coronavirus, and a vaccine containing the RNA. The mRNA or vaccine of the present application has one or more of the following characteristics: (1) higher protein expression, (2) capable of stimulating a stronger immune response, (3) producing more cytokines, (4) having Reduced immunogenicity, and (5) production of higher levels of neutralizing antibodies.

On the one hand, the present application provides an RNA encoding the new coronavirus S protein, wherein the new coronavirus S protein includes the amino acid sequence shown in SEQ ID NO.5 or SEQ ID NO.6. In certain embodiments, the amino acid sequence of the novel coronavirus S protein is shown in SEQ ID NO.5 or SEQ ID NO.6. In certain embodiments, the S protein comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, Amino acid sequences with at least 97%, at least 98% or at least 99% sequence identity.

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 the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2. In certain embodiments, the nucleotide sequence of the RNA is shown in SEQ ID NO. 1 or SEQ ID NO. 2. In certain embodiments, the sequence of the RNA has at least 80%, at least 85%, at least 90%, at least 95%, or at least 96% similarity with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2. %, at least 97%, at least 98%, or at least 99% sequence identity.

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 contains more than 30 adenosines.

In certain embodiments, in the RNA, from the 5' end to the 3' end, the structure of the poly(A) tail is: 30 adenylate, a connecting sequence and 70 adenylate. For example, the poly(A) tail may be 100-200 nucleotides in length, such as about 120 nucleotides in length. For example, about 110 nucleotides.

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

In certain embodiments, the poly(A) tail of the RNA comprises the nucleotide sequence set forth in SEQ ID NO. 9. 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% similarity with the nucleotide sequence shown in SEQ ID NO.9 %, at least 98%, or at least 99% sequence identity.

In certain embodiments, the RNA comprises the nucleotide sequence set forth in SEQ ID NO. 10 or SEQ ID NO. 11. In certain embodiments, the sequence of the RNA has at least 80%, at least 85%, at least 90%, at least 95%, or at least 96% similarity with the nucleotide sequence shown in SEQ ID NO.10 or SEQ ID NO.11. %, at least 97%, at least 98%, or at least 99% sequence identity.

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

In certain embodiments, the RNA comprises modifications at one or more positions selected from the group consisting of: 5' Cap structure, 5' untranslated sequence, open reading frame, 3' untranslated sequence, and 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, axonemal dynein, or a homolog, fragment or variant thereof 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, or a homolog, fragment or variant thereof, Tyrosine hydroxylase gene, heat shock protein 70 gene, lipoxygenase gene and collagen alpha gene.

In certain embodiments, the chemical structural formulas of the first and second structural units in the 5'Cap structure are as follows:

In certain embodiments, the modified nucleotides of the RNA comprise one or more nucleotides selected from the group consisting of: N1-Methylpseudo-UTP ), pseudouridine triphosphate (pseudo-UTP), 5-methoxyuridine triphosphate (5-Methoxy-UDP) and 5-methylcytidine triphosphate (5-Methyl-CTP).

In certain embodiments, all the structural unit U bases in the RNA sequence are replaced by m1Ψ=1-methyl-3'-pseudouridylyl.

On the other hand, the present application provides a DNA encoding the new coronavirus S protein, wherein the new coronavirus S protein includes the amino acid sequence shown in any one of SEQ ID NO. 5-6.

In another aspect, the present application provides a DNA transcribing the RNA described in the present application.

In certain embodiments, the DNA is codon optimized. In certain embodiments, the DNA comprises the nucleotide sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8. In certain embodiments, the nucleotide sequence of the DNA is shown in SEQ ID NO.7 or SEQ ID NO.8. In some embodiments, the DNA sequence has at least 80%, at least 85%, at least 90%, at least 95%, or at least 96% similarity with the nucleotide sequence shown in SEQ ID NO.7 or SEQ ID NO.8. %, at least 97%, at least 98%, or at least 99% sequence identity.

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

In certain embodiments, the DNA comprises the nucleotide sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4. In certain embodiments, the nucleotide sequence of the DNA is shown in SEQ ID NO.3 or SEQ ID NO.4. In some embodiments, the DNA sequence has at least 80%, at least 85%, at least 90%, at least 95%, or at least 96% similarity with the nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.4. %, at least 97%, at least 98%, or at least 99% sequence identity.

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

Regarding the specific mode of delivery, there is no particular limitation, and those conventionally used in the art can be adopted. For example, reference can be made to the methods disclosed in published patents US20160376224A1 or WO2015199952A1.

The delivery vehicle in the compositions described herein may be lipid nanoparticles. The nanolipid particles may comprise one or more (eg 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 (eg, physiological pH). The cationic lipids may include, but are not limited to, SM102, 3-(didodecylamino)-N1,N1,4-triacontyl-1-piperazineethylamine (KL10), N1-[2-(Docosylamino)ethyl]-N1,N4,N4-Tridodecyl-1,4-piperazinediethylamine (KL22), 14,25-tricosane Base-15,18,21,24-tetraazaoctaporanane (KL25), DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, Octyl-CLinDMA, octyl-CLinDMA (2S), DODAC , DOTMA, DDAB, DOTAP, DOTAP.C1, DC-Choi, DOSPA, DOGS, DODAP, DODMA and DMRIE. In certain embodiments, the molar proportion of the cationic lipid in the lipid nanoparticle is about 40-70%, for example, about 40-65%, about 40-60%, about 45-55%, or about 48 -53%. In this application, the nanolipid particles may comprise one or more (eg 1, 2, 3, 4, 5, 6, 7 or 8) non-cationic lipids. The non-cationic lipids may include anionic lipids. Anionic lipids suitable for lipid nanoparticles of the present application may include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-lauroylphosphatidylethanolamine, N-succinylphosphatidylethanolamine , N-glutarylphosphatidylphosphoethanol group, and other neutral lipids to which anionic groups are attached.

The non-cationic lipids may include neutral lipids. Neutral lipids suitable for lipid nanoparticles of the present application may include phospholipids, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine ( DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl- Phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-trans Formula PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof. Additionally, lipids having a mixture of saturated and unsaturated fatty acid chains can be used. For example, the neutral lipids described herein may be selected from DOPE, DSPC, DPPC, POPC or any related phosphatidylcholines.

In certain embodiments, the molar proportion of the phospholipids in the lipid nanoparticles is about 5-20%. In the present application, the nanolipid particles may comprise lipid conjugates, for example, polyethylene glycol (PEG) modified lipids and derivatized lipids. PEG-modified lipids may include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently linked to lipids having alkyl chains of C6-C20 length. The addition of these components can prevent lipid aggregation, increase circulation duration, facilitate delivery of lipid-nucleic acid compositions to target cells, or rapidly release nucleic acids. For example, the polyethylene glycol (PEG) modified lipid molecule can be a PEG-ceramide with a shorter acyl chain (eg, C14 or C18). In certain embodiments, the molar proportion of polyethylene glycol (PEG)-modified lipid molecules in the lipid nanoparticles is about 0.5-2%, for example, 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 this application, the nanolipid particles may also contain cholesterol. In certain embodiments, the molar proportion of cholesterol in the lipid nanoparticle is about 30-50%, for example, about 35-45%.

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

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

On the other hand, the present application provides a composition, which may include the mRNA described in the present application and may also include a delivery carrier.

In certain embodiments, the delivery vehicle includes liposomes.

In certain embodiments, the delivery vehicle includes lipid nanoparticles (LNP).

In certain embodiments, the lipid nanoparticles include cationic lipids, non-cationic lipids, and cholesterol.

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

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

On the other hand, the present application provides a method for preparing RNA vaccines, which method includes: a. dissolving ionized lipids, PEGylated lipids, cholesterol and derivatives and phospholipids in an ethanol solution; b. Mix the lipid ethanol solution and the RNA aqueous solution through a microfluidic mixer to obtain lipid nanoparticles (LNP); c. Separate and purify the LNP obtained in step b to obtain the RNA vaccine, wherein the RNA is as follows Application as defined.

In another aspect, the present application provides a pharmaceutical composition comprising the RNA, the DNA, the composition, the liposome nanoparticle formulation, the vaccine, and/or a pharmaceutically acceptable composition thereof Carrier.

On the other hand, the present application provides a method for treating and/or preventing diseases or conditions related to novel coronavirus infection, the method comprising administering the RNA, the DNA, the composition, The liposomal nanoparticle formulation, the vaccine, and/or the pharmaceutical composition.

In some embodiments, the pharmaceutical composition of the present application can be administered to a subject by any method known to those skilled in the art, such as parenterally, orally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneous, intraperitoneal, intraventricular, intracranial, intravaginal, or intratumoral.

In the present application, the RNA, the DNA, the vector, the cell, the composition, the liposome nanoparticle formulation, the pharmaceutical composition and/or the vaccine can be administered in a single application, multiple applications or continuous application, And all are safe. After the initial administration, subsequent administration of the composition and/or pharmaceutical composition may shorten the onset of action. In this application, the RNA, the DNA, the vector, the cell, the composition, the liposome nanoparticle formulation, the pharmaceutical composition and/or the vaccine may be combined with other active Co-administration of substances or therapeutic/preventive ingredients. In this application, the RNA, the DNA, the vector, the cell, the composition, the liposome nanoparticle formulation, the pharmaceutical composition and/or the vaccine may be used in other active Before or after the substance or therapeutic/preventive ingredient is administered.

On the other hand, the present application provides the use of the RNA, the DNA, the composition, the liposome nanoparticle preparation, the vaccine, and/or the pharmaceutical composition in the preparation of medicines, The above-mentioned drugs are used to treat and/or prevent diseases or conditions related to novel coronavirus infection.

In certain embodiments, the disease or condition associated with novel coronavirus infection includes pneumonia, such as COVID-19.

In certain embodiments, the drugs (eg, the vaccines) can be administered sequentially.

In certain embodiments, the drug (eg, the vaccine) can be administered multiple times, eg, two, three, four, or more times.

In some embodiments, the drug (eg, the vaccine) can be administered sequentially with other vaccines against the novel coronavirus. Other vaccines against the new coronavirus can be any other vaccines in the existing technology that prevent the new coronavirus, including but not limited to inactivated vaccines, mRNA vaccines, DNA vaccines, recombinant protein vaccines and/or adenovirus vaccines. For example, the medicine of the present application (for example, the vaccine) can be administered first, and then other vaccines against the new coronavirus can be administered, or other vaccines against the new coronavirus can be administered first, and then the medicine of the present application (for example, the vaccine) can be administered vaccine).

In certain embodiments, when the drugs (eg, the vaccine) are administered sequentially, the time interval between two adjacent 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 more . The specific time can be determined based on the subject's constitution, health status, immune response level after the first vaccine administration, etc.

On the other hand, the present application provides a vector, which includes the RNA and the DNA.

For example, the vector may be a viral vector, such as an adenoviral vector, an adeno-associated viral vector, and/or a lentiviral vector.

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

In certain embodiments, the vector comprises a viral vector.

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

On the other hand, the present application provides a method of producing antibodies against the new coronavirus, which includes administering the RNA, the DNA, the composition, the liposome nanoparticle formulation, the vaccine, and/ or the pharmaceutical composition.

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

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

Compared with the prior art, the beneficial effects of the present invention are:

1. Both types of mRNA provided by the present invention can be translated in cells and produce high levels of S protein of the new coronavirus Delta strain. Each mRNA is injected into mice through a preparation formed after encapsulation with liposome nanoparticles. Induces mice to produce high titers of neutralizing antibodies. Compared with other mRNA or mRNA without codon optimization, the mRNA of the present application can stimulate a stronger immune response and produce higher levels of neutralizing antibodies.

2. The two mRNAs provided by the present invention are CVG024 and CVG025. Both mRNAs contain open reading frames encoding the full length of the new coronavirus S protein. The open reading frames of both mRNAs have been codon-optimized. The full-length sequence of CVG024 is SEQ ID NO.3, and the full-length sequence of CVG025 is SEQ ID NO.4. As shown, the encoded protein sequences are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively. Compared with the S protein of the Wuhan-Hu-1 strain, there are mutations at multiple sites in the S protein of the Delta strain. The protein sequences SEQ ID NO.5 and SEQ ID NO.6 expressed by the mRNA of the present invention both contain multiple mutations that appear in the S protein of the new coronavirus Delta strain, such as T19R, T95I, G142D, E156G, DEL157/158, L452R , T478K, D614G, P681R, D950N; the protein sequence SEQ ID NO.6 also contains the K417N mutation that appears in the S protein of the Delta strain. Both mRNAs contain a 5'Cap structure, a 5'untranslated sequence (5'UTR), a 3'untranslated sequence (3'UTR) and a poly(A) tail. The structural unit U bases in both mRNA sequences are all replaced by m1Ψ (1-methyl-3'-pseudouridylyl).

Description of the drawings

Figure 1 shows the expression results of mRNA in HEK 293T/17 cells in Example 2.

Figure 2 shows Western Blot detection of translation products after mRNA transfection into HEK 293T/17 cells in Example 2.

Figure 3 shows the particle size and uniformity of the mRNA-LNP coated with CVG025 mRNA in Example 3 at room temperature (RT) or after thawing at -80°C.

Figure 4 shows the encapsulation rate of the mRNA-LNP coated with CVG025 mRNA in Example 3 at room temperature (RT) or after thawing at -80°C.

Figure 5 shows the surface potential properties of the mRNA-LNP coated with CVG025 mRNA in Example 3 at room temperature (RT) or after thawing at -80°C.

Figure 6 shows the neutralizing antibody titers against Delta pseudovirus in mouse serum one week after the initial immunization in Example 4; the abscissas from left to right correspond to Saline, CVG025 1ug/mouse, CVG025 10ug/mouse, and CVG025 25ug/piece.

Figure 7 shows the neutralizing antibody titer against Delta pseudovirus in mouse serum one week after two immunizations in Example 4, where WT refers to the SARS-CoV-2 wild strain; the abscissa corresponds from left to right The order is Saline, CVG0251μg/bird, CVG025 10μg/bird, CVG025 25μg/bird.

Figure 8 shows the neutralizing antibody titer against Delta pseudovirus in mouse serum two weeks after two immunizations in Example 4, where WT refers to the SARS-CoV-2 wild strain; the abscissa is from left to right The corresponding ones are Saline, CVG0251μg/piece, CVG025 10μg/piece, and CVG025 25μg/piece.

Figure 9 shows that neutralizing antibodies induced by CVG025 can cross-react with Omicron BA.2 subtype.

Figure 10 shows the antigen-specific responses caused by CVG025 in CD4 ⁺ T cells (Figure 10A-Figure 10D) and CD8 ⁺ T (Figure 10E-Figure 10H) cells, in which wild type (Figure 10A and Figure 10E), Delta (Figure 10B and Figure 10F) or Omicron (Figure 10C and Figure 10G). The number of IFNγ ⁺ /CD69 ⁺ T cells increased after treatment with Spike protein, and IL2+ lymphocytes significantly increased in mouse splenocytes stimulated by delta S protein (Figure 10I). At the same time, IL-5 was not elevated (Fig. 10J).

Figure 11 shows the serological evaluation and T cell immunity of mice boosted with CVG025 after two doses of SARS-Covid-2 inactivated vaccine. A: Schematic diagram of the mRNA vaccine boosting immunization protocol. B: Mice boosted with CVG025 with pseudovirus. Neutralization test to measure neutralizing antibody titers, CH: Splenocytes were collected 4 weeks after the secondary immunization and stimulated with different variants of S protein for 48 hours, then stimulated with CVG025 and then used flow cytometry to measure CD69 in CD4 and CD8 T cells. ⁺ Ratio of T cells, IJ: Ratio of Tfh (Foxp3-PD1+) and DC (CD11C+IA/IE+) in the spleen.

Detailed ways

The implementation of the invention of the present application will be described below with specific examples. Those skilled in the art can easily understand other advantages and effects of the invention of the present application from the contents disclosed in this specification.

In this application, the term "delivery vector" generally refers to a transfer vehicle capable of delivering an agent (eg, mRNA) to a target cell. Delivery vehicles can deliver agents (eg, mRNA) to specific cell subtypes. For example, by means of inherent characteristics of the delivery vehicle or by means of a moiety coupled to, contained within (or a moiety bound to the carrier) such that the moiety and the delivery vehicle are maintained together, thereby rendering the moiety sufficient to target the target. Targeting the delivery vector to certain types of cells. The delivery vehicle may also increase the in vivo half-life of the agent to be delivered (eg, mRNA) and/or the bioavailability of the agent to be delivered. Delivery vectors 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 delivery vector has properties (e.g., size, charge, and/or pH) that are effective in delivering the delivery vector and/or the molecule entrapped therein (e.g., mRNA) to the target. cells, reduce immune clearance and/or promote retention in that target cell.

In this application, the term "DNA" generally includes cDNA or genomic DNA, and "RNA" generally includes mRNA, and also includes DNA produced using nucleotide analogs (such as peptide nucleic acids and non-naturally occurring nucleotide analogs) or RNA analogs, and hybrids thereof. DNA or RNA can be single-stranded or double-stranded. In this application, the term "mRNA" generally refers to an RNA transcript that has been processed to remove introns and capable of being translated into a polypeptide.

In this application, the term "modification" when applied to a nucleic acid, such as RNA or DNA, generally means that the nucleic acid has a different nucleotide molecule, a different nucleotide sequence, compared to the corresponding wild type, consisting of Different bond compositions and/or the incorporation of unnatural moieties into their structure. For example, the modification may include modification of a nucleotide, for example, the nucleotide may include a modified base, sugar, or phosphate group. For example, the modifications may include polypeptides or proteins with different nucleotide sequences but encoding the same amino acid sequence, or the same function. The modification may be chemical modification and/or biological modification. "Chemical modification" may include modifications that introduce chemicals different from those found in wild-type or naturally occurring nucleic acids, e.g., covalent modifications, e.g., the introduction of modified nucleotides (e.g., nucleotide analogs , or introduce side groups not naturally found in these nucleic acid molecules). The term "modified nucleotide" generally refers to units in nucleic acid polymers that contain modified bases, sugars, or phosphate groups, or that incorporate non-natural moieties into their structure.

According to the present invention, naturally occurring or modified nucleosides and/or nucleotides, or optimized nucleotides, may be used to prepare modified nucleic acids, such as modified mRNA. For example, 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-deazaadenosine , 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, (for example, N-1-methyl-pseudouridine) , 2-thiouridine and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methyl sylated bases); inserted bases; modified sugars (e.g., 2'-fluoribose, ribose, 2'-deoxyribose, arabinose, and hexose); modified phosphate groups (e.g., phosphorothioate and 5 '-N-phosphoramidite bond), or any combination thereof. These modified nucleotides can be natural nucleotides, or artificially optimized or improved nucleotides.

RNA molecules (eg, mRNA) can include at least 0.1% modified nucleotides. The fraction of modified nucleotides can be calculated as: number of modified nucleotides/total number of nucleotides × 100%. In some embodiments, the RNA molecule includes about 0.1% modified nucleotides to about 100% modified nucleotides. In some embodiments, the RNA molecule includes about 0.1% modified nucleotides, about 0.2% modified nucleotides, about 0.5% modified nucleotides, about 1% modified nucleotides, about 2% modified nucleotides, about 5% modified nucleotides, about 10% modified nucleotides, about 20% modified nucleotides, about 50% modified nucleotides, or about 100% modified nucleotides.

As used herein, the term "codon optimization", when applied to nucleic acids, generally means the replacement of one, at least one, of the parent polypeptide-encoding nucleic acid with a codon encoding the same amino acid residue that has a different relative frequency of use in the cell. , or a nucleic acid encoding a polypeptide in which one or more codons have been modified to have improved expression in a cell, such as a mammalian cell or a bacterial cell.

In this application, the term "pharmaceutical composition" generally refers to a preparation in a form that is effective in allowing the biological activity of the active ingredient (eg, vaccine, composition, pharmaceutical composition, vector, cell, DNA or RNA of the present application) , and it does not contain additional ingredients that would be unacceptably toxic to the subject to whom the formulation is to be administered. These preparations can be sterile.

In this application, the term "S protein", which may also be called "Spike protein" or "Spike protein", generally refers to the membrane protein on the surface of coronavirus, which can form protruding homotrimers on the surface of the virus.

As used herein, 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 this application, the term "lipid nanoparticle" generally refers to particles that contain multiple (ie, more than one) lipid molecules physically bound to each other (eg, covalently or non-covalently) by intermolecular forces. Lipid nanoparticles can be, for example, microspheres (including unilamellar and multilamellar vesicles, such as liposomes), the dispersed phase in emulsions, micelles or the internal phase in suspensions. Lipid nanoparticles can include one or more lipids (eg, cationic lipids, noncationic lipids, and PEG-modified lipids).

In this application, the term "liposome" generally refers to a vesicle having an internal space separated from an external medium by a membrane of one or more bilayers. For example, the membrane of the bilayer can be formed by amphipathic molecules, such as synthetic or naturally derived lipids containing spatially separated hydrophilic and hydrophobic domains; as another example, the membrane of the bilayer can be formed by amphiphilic molecules. Polymer and surfactant formation.

In this application, the term "new coronavirus" generally refers to severe acute respiratory syndrome coronavirus 2, the full English name is Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 belongs to the Betacoronavirus subgenus of the Coronaviridae family and the Sarbecovirus subgenus. SARS-CoV-2 is an enveloped, non-segmented, positive-stranded single-stranded RNA virus. It can cause novel coronavirus pneumonia (COVID-19).

In this application, the term "pharmaceutically acceptable carrier" generally refers to any solvent, dispersion medium, coating, isotonic and absorption delaying agents and other adjuvants, excipients or Other drug carriers.

In this application, the term "carrier" is intended to include any solvent, dispersion medium, coating, diluent, buffer, pharmaceutically acceptable for administration to the relevant animal or, if applicable, acceptable for therapeutic or diagnostic purposes. Isotonic agents, solutions, suspensions, colloids, inert bodies, etc., or combinations thereof.

As used herein, the term "effective amount" refers to an amount capable of treating or ameliorating a disease or condition or producing the 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. When a first nucleic acid or amino acid sequence has an identical primary sequence to a second nucleic acid or amino acid sequence, the word "identity" may be substituted for the word "homology." 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 evaluate whether a given sequence has identity or homology with another selected sequence.

In the context of two or more nucleic acid or polypeptide sequences, the term "identity" or "percent identity" refers to the meaning when using one of the sequence comparison algorithms described below (or other algorithms available to one of ordinary skill) or by visual A check measures comparison and alignment for maximum correspondence when two or more sequences or subsequences are identical or have a specific percentage of identical amino acid residues or nucleotides.

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

In this article, the meaning of "SEQ ID NO.1-11" is as shown in the following table:

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 reagents, components, or pharmaceutically formulated compositions of the invention. Optionally, such kits may include one or more sets of instructions for use of the encapsulated compositions, for example, in laboratory or clinical applications.

As used herein, the terms "prevention" or "treatment" refer to the administration of a compound, alone or included in a pharmaceutical composition, prior to the onset of clinical symptoms of a disease state, to prevent any symptom, aspect or feature of the disease state. Such prevention and suppression need not be absolutely considered medically useful.

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

1. RNA encoding the new coronavirus S protein, wherein the new coronavirus S protein includes the amino acid sequence shown in SEQ ID NO.5 or SEQ ID NO.6.

2. The RNA according to embodiment 1, which is mRNA.

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

4. The RNA of any one of embodiments 1-3, which comprises the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

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

6. The RNA of embodiment 5, wherein the poly(A) tail contains more than 30 adenosines.

7. The RNA according to embodiment 5 or 6, wherein, from the 5' end to the 3' end, the structure of the poly(A) tail is: 30 adenylic acid, a connecting sequence and 70 adenylic acid.

8. The RNA of any one of embodiments 5-7, wherein the poly(A) tail comprises the nucleotide sequence shown in SEQ ID NO.9.

9. The RNA of any one of embodiments 1-8, which comprises the nucleotide sequence shown in SEQ ID NO.10 or SEQ ID NO.11.

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 modifications at one or more positions selected from the group consisting of: 5' Cap structure, 5' untranslated sequence, open reading frame, 3' Untranslated sequences and poly(A) tails.

12. The RNA of embodiment 10 or 11, wherein the modified nucleotides comprise one or more nucleotides selected from the group consisting of N1-methylpseudouridine triphosphate (N1- Methylpseudo-UTP), pseudouridine triphosphate (pseudo-UTP), 5-methoxyuridine triphosphate (5-Methoxy-UDP) and 5-methylcytidine triphosphate (5-Methyl-CTP).

13. DNA transcribing the RNA of any one of embodiments 1-12, the DNA comprising the nucleotide sequence shown in SEQ ID NO.7 or SEQ ID NO.8.

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 10-14, which comprises the nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

16. A composition comprising the RNA of any one of embodiments 1-9, and a delivery vehicle.

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

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

19. The composition of any one of embodiments 16-18, wherein the lipid nanoparticles comprise cationic lipids, non-cationic lipids and cholesterol.

20. A liposome nanoparticle formulation coated with the RNA of any one of embodiments 1-12.

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

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

23. A method for treating and/or preventing diseases or conditions related to novel coronavirus infection, the method comprising administering the RNA described in any one of embodiments 1-12, embodiments 13-15 to a patient in need The DNA described in any one of embodiments 16-19, the liposome nanoparticle preparation described in embodiment 20, the vaccine described in embodiment 21, and/or the embodiment The pharmaceutical composition described in 22.

24. The RNA described in any one of embodiments 1-12, the DNA described in any one of embodiments 13-15, the composition described in any one of embodiments 16-19, or the composition described in embodiment 20 The liposome nanoparticle preparation, the vaccine described in Embodiment 21, and/or the use of the pharmaceutical composition described in Embodiment 22 in the preparation of medicines for treating and/or preventing novel coronavirus infection-related disease or illness.

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

29. A method for producing antibodies against novel coronavirus, comprising administering the RNA described in any one of embodiments 1-12, the DNA described in any one of embodiments 13-15, or any one of embodiments 16-19 The composition described in Item 2, the liposome nanoparticle preparation described in Embodiment 20, the vaccine described in Embodiment 21, and/or the pharmaceutical composition described in Embodiment 22.

30. A method of activating immunity, comprising administering the RNA described in any one of embodiments 1-12, the DNA described in any one of embodiments 13-15, or the DNA described in any one of embodiments 16-19 The composition, 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.

Example

The technical solution of the present invention will be described in detail below with reference to examples. The reagents and biological materials used below are all commercial products unless otherwise specified.

The specific experimental process of the present invention is: 1. Obtain the gene sequence of the SARS-CoV-2 virus; 2. Obtain the protein sequence of the SARS-CoV-2 virus; 3. Design the DNA template sequence encoding the antigenic protein; 4. In vitro transcription production mRNA; 5. liposome nanoparticles encapsulate mRNA; 6. immunize mice with liposome nanoparticles encapsulating mRNA; 7. detect neutralizing antibodies in mouse serum.

Example 1 Preparation of mRNA

After the RNA sequence of the present invention is optimized, experimental methods known to those skilled in the art are used to clone the DNA template of the RNA in vitro transcription into the pUC57 vector, transform it into E. coli competent cells, and preserve the positive cloned strains. Expand the culture strain, extract and purify the DNA template plasmid, linearize the plasmid after digestion, use the linearized plasmid as a template to perform RNA in vitro transcription (IVT), and purify the RNA product. Specific steps as follows:

The company was commissioned to synthesize two gene sequences encoding the full length of the S protein of the new coronavirus. The gene sequences have been codon-optimized. The sequences are as shown in SEQ ID NO.7 and SEQ ID NO.8, and the translated protein sequences are as SEQ ID NO.5. , SEQ ID NO.6. Preferably, the two genes are synthesized together with the 5'UTR DNA sequence, the 3'UTR DNA sequence, and the poly(A) tail. Clone the synthetic gene product (sequence shown in SEQ ID NO.3 and SEQ ID NO.4) containing 5'UTR, open reading frame, 3'UTR, and poly(A) tail into the pUC57 vector. After the vector is amplified, it is digested with restriction endonuclease to obtain the linearized vector. Use the purified linearized vector as a template for RNA in vitro transcription, add T7 RNA polymerase, CTP, GTP, ATP, m1Ψ (1-methyl-3'-pseudouridylyl) modified UTP (i.e. N1-methylpseudouridine triphosphate (N1-Methylpseudo-UTP)), Cap analog ((3'-OMe-m7G)(5')ppp(5')(2'-OMeA)pG, that is, the 3' hydroxyl group of m7G has been methylated , ensuring high translation efficiency and anti-reverse transcription effect), and other necessary components known to those skilled in the art, incubate at 37°C for 1-5 hours. After the reaction is completed, digest with DNase to remove DNA and further purify to obtain the mRNA product. The mRNA concentration and integrity are measured by methods well known to those skilled in the art. Two kinds of mRNA were prepared, named CVG024 and CVG025, and the nucleic acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

Example 2 Detection of mRNA cell translatability

Prepare CVG025 mRNA according to the steps described in Example 1, refer to the transfection reagent product instructions, mix CVG025 mRNA and transfection reagent according to the guided ratio, transfect into HEK 293T/17 cells, and detect the expression of the mRNA after transfection of the cells, as shown in the figure As shown in 1, Figure 1 shows the expression of the mRNA in HEK 293T/17 cells. The full-length expression of the new coronavirus S protein was detected by Western Blot. The detected S protein results are shown in Figure 2. Spike in Figure 2 indicates the molecular weight of the new coronavirus S protein, Neg ctrl.#1 and Neg ctrl.#2 are negative control groups, and no expression of the new coronavirus S protein was observed; Positive ctrl.#1 and Positive ctrl.# 2 is a positive control, observing the expression of the new coronavirus S protein; CVG025mRNA#1-#4 are four groups of independently prepared samples, and all four groups of samples can express protein products with the same molecular weight as the new coronavirus S protein. The results in Figures 1 and 2 show that CVG025 mRNA can be translated into the S protein of the new coronavirus.

Example 3 Liposomal Nanoparticles mRNA

The mRNA obtained in Example 1 was encapsulated: cationic lipid (MC3), DSPC, cholesterol and PEG-lipid were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. Lipid nanoparticles (LNPs) are prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. Briefly, the mRNA and four components of Example 1 The lipid mixture was made into mRNA-LNPs using a microfluidic nanoprecipitation process in which an aqueous solution of mRNA at acidic pH is rapidly mixed with an ethanolic solution of lipids. The ethanol in the crude product was then removed by tangential flow ultrafiltration (TFF), followed by buffer exchange using PBS solution (1x, pH 7.4). Next, use sucrose solution to dilute the mRNA in the neutralized product to 0.5 mg/mL. Finally, the product is sterile filtered through a 0.22 μm Sartopore PES membrane, and aliquots are subsequently stored at room temperature or frozen at -80°C (1.0 mL fill).

Characterization of mRNA-LNP: Particle size, polydispersity (PDI) and zeta potential of mRNA-LNP were measured by dynamic light scattering (Malvern Nano ZS Zetasizer). The diameter is characterized by Z average. The encapsulation efficiency (EE) of mRNA in LNP is defined as the mass ratio of encapsulated mRNA/total mRNA in the final mRNA-LNP product. Using specific intercalating fluorescent dyes (Quant-iT ^TM RNA Reagent) quantitatively detects nucleic acids such as mRNA. Calculate the mRNA concentration based on the calibration curve generated using the mRNA standards. The relative proportions of encapsulated mRNA in mRNA-LNPs were determined by the ratio of the fluorescence signal in the absence to presence of surfactant that disperses the LNPs. The signal in the absence of surfactant indicates the level of free mRNA, while the signal in the presence of surfactant serves as a measure of the total mRNA in the sample.

The particle size and uniformity of mRNA-LNP coated with CVG025 mRNA at room temperature (RT) and after freezing and thawing at -80°C are shown in Figure 3. The LNP diameter in both cases is between 80-100nm, based on PDI Measuring the dispersion of LNP particle diameters, the PDI values are all low, indicating that the size of the mRNA-LNP of the present invention is relatively uniform. The mRNA encapsulation rate of the mRNA-LNP coated with CVG025 mRNA reached almost 100% at room temperature (RT) and after freezing and thawing at -80°C, as shown in Figure 4, indicating that the present invention uses LNP to prepare the mRNA COVID-19 vaccine. The preparation can encapsulate mRNA in LNP very efficiently and is still very stable after freezing and thawing at -80°C. The nanoparticle surface potential properties of the mRNA-LNP coated with CVG025 mRNA maintained negative charge properties at room temperature (RT) and after freezing and thawing at -80°C, as shown in Figure 5, indicating that the mRNA-LNP of the present invention can be uniformly suspended. Distributed in the solution; the absolute value of the charge is small, indicating that the mRNA-LNP can still pass through the cell membrane that also has a negative charge on the outer surface.

Example 4 Animal Immunization

After filtering the liposome nanoparticle preparation coated with CVG025 mRNA in Example 3, 6-8 week old female Balb/c mice were injected. The control group was injected with physiological saline (Saline). The injection dose of the liposome nanoparticle preparation was 1 μg. /bird, 10μg/bird, 25μg/bird. Seven days after the first immunization, the blood of mice was collected, and the neutralizing antibody titer against Delta pseudovirus or wild-type new coronavirus pseudovirus in the serum was detected. The results are shown in Figure 6. On the 21st day after the initial immunization, the mice were injected again with the filtered liposome nanoparticle preparation of Example 3. This is secondary immunity. One week after the secondary immunization, the blood of the mice was collected and the neutralizing antibody titer in the serum was detected. The results are shown in Figure 7. Two weeks after the secondary immunization, the blood of the mice was collected and the neutralizing antibody titer in the serum was detected. The results are shown in Figure 8.

Experimental results show that when liposome nanoparticles made of CVG025 mRNA and liposome components provided by the invention are injected into mice, they induce the production of high-level neutralizing antibodies against wild-type and mutant new coronavirus strains. And the level of neutralizing antibodies increased with the increase in liposome nanoparticle concentration, and the titer in the Delta mutant strain group was higher.

CVG024mRNA, whose nucleic acid sequence is shown in SEQ ID NO.1, has similar immune effects to CVG025mRNA. Both mRNAs can be translated in cells and produce high levels of new coronavirus S protein. Each mRNA is transported through liposome nanoparticles The encapsulated preparation is injected into mice to induce the mice to produce high-titer neutralizing antibodies. Compared with other mRNAs or mRNAs without codon optimization, the two mRNAs of this application can stimulate stronger immune responses and produce higher levels of neutralizing antibodies.

Example 5 Cross-reaction of neutralizing antibodies induced by CVG025 with Omicron subspecies

Mice were immunized twice with CVG025 on days 0 and 14, and serum samples from animals 14 days after the second immunization were incubated with Omicron BA.1 and BA.2 HIV pseudoviruses and tested for their neutralizing activity. The results (Figure 9) showed that the antibodies induced by CVG025 cross-reacted with Omicron BA.1 and BA.2, and the levels of BA.1 and BA.2 were similar.

Example 6 CVG025 induces cellular immunity

This example detects the T cell immune response of CVG025 immunized animals. Intracellular staining was performed using cytokines induced by restimulation of different variants of full-length S protein to evaluate the S protein-specific cellular immune function. The inoculation method was the same as above, and spleen cells were collected for analysis 4 weeks after the second inoculation. The results (Fig. 10) showed that CVG025 can induce antigen-specific responses of CD8 and CD4 T cells, which can be seen from the increase in the number of IFNγ ⁺ /CD69 ⁺ T cells after treatment with wild-type, Delta or Omicron's Spike protein, respectively (Fig. 10A-Figure 10H). The results showed that mice immunized with CVG025 produced a strong immune response to the new coronavirus. The ELSA method to detect the expression of IL2 in spleen cells of mice stimulated by S protein also showed a significant increase in IL2+ lymphocytes in mice vaccinated with CVG025 (Figure 10I). At the same time, IL-5 was not increased in all samples (Figure 10J), indicating that CVG025 mainly induces a specific Th1 immune response rather than Th2.

Example 7 CVG025 Booster Immunization

First, the BBIBP-CorV vaccine prepared with inactivated wild virus (2 doses, China Pharmaceutical Group Beijing Biotechnology Co., Ltd. Product Research Institute) immunized Balb/C mice, and then the Balb/C mice were vaccinated with the third booster shot of CVG025. Similarly, Balb/C mice in the control group were vaccinated with the third BIBP-CorV homologous booster shot.

The results are shown in Figure 11. After two doses of BBIBP-CorV immunization, one dose of CVG025 can induce the production of neutralizing antibodies, and antibodies can be produced in three strains (WT, delta, omicron), increasing CD69 ⁺ T The proportion of cells enhances T cell immunity.

The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

Claims (15)

1. RNA encoding the new coronavirus S protein, wherein the new coronavirus S protein includes the amino acid sequence shown in SEQ ID NO.5 or SEQ ID NO.6.
2. The RNA of claim 1, comprising the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.
3. The RNA of any one of claims 1-2, comprising the following group of structures: 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 shown in SEQ ID NO.9.
5. The RNA of any one of claims 1-4, which includes the nucleotide sequence shown in SEQ ID NO. 10 or SEQ ID NO. 11.
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 nucleotides comprise one or more nucleotides selected from the group consisting of N1-methylpseudouridine triphosphate (N1-Methylpseudo-UTP) , pseudouridine triphosphate (pseudo-UTP), 5-methoxyuridine triphosphate (5-Methoxy-UDP) and 5-methylcytidine triphosphate (5-Methyl-CTP).
8. DNA transcribing the RNA of any one of claims 1-7, said DNA comprising the nucleotide sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8.
9. The DNA of claim 8, wherein the DNA further comprises the following group of structures: a 5' untranslated sequence, a 3' untranslated sequence and a poly(A) tail.
10. The DNA of any one of claims 8-9, which includes the nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.4.
11. A liposome nanoparticle preparation coated with the RNA described in any one of claims 1-7.
12. A vaccine comprising the RNA described in any one of claims 1-7, the DNA described in any one of claims 8-10, and/or the lipid nanoparticle preparation described in claim 11.
13. A pharmaceutical composition comprising the RNA described in any one of claims 1-7, the DNA described in any one of claims 8-10, and/or the lipid nanoparticle preparation described in claim 11 , the vaccine according to claim 12, and/or its pharmaceutically acceptable carrier.
14. The RNA according to any one of claims 1 to 7, the DNA according to any one of claims 8 to 10, and/or the lipid nanoparticle preparation according to claim 11, and the vaccine according to claim 12 , and/or claim 13 The use of a pharmaceutical composition in the preparation of medicaments for treating and/or preventing diseases or conditions related to novel coronavirus infection.
15. According to the use of claim 14, the medicament is suitable for sequential administration.