CN116768987A - mRNA vaccine encoding novel coronavirus S protein - Google Patents
mRNA vaccine encoding novel coronavirus S protein Download PDFInfo
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- CN116768987A CN116768987A CN202210224642.4A CN202210224642A CN116768987A CN 116768987 A CN116768987 A CN 116768987A CN 202210224642 A CN202210224642 A CN 202210224642A CN 116768987 A CN116768987 A CN 116768987A
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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. 5-6. The mRNA comprises a nucleotide sequence shown in any one of SEQ ID NO.1 and SEQ ID NO. 2. The two 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
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, 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. Three highly pathogenic coronaviruses, SARS-CoV, MERS-CoV and SARS-CoV-2, have emerged into the twenty-first century and pose a serious threat to human health. The global mortality rate of SARS-CoV reaches 10% and the global mortality rate of MERS-CoV reaches 35%. Development of vaccines against novel coronaviruses is an urgent need to suppress the pandemic of novel coronaviruses.
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, as the time of infection in the population increases, various mutant strains of SARS-CoV-2 have been detected, such as Alpha, beta, gamma, delta, omicron strains. Compared with wild strains, the mutant strains have stronger infection capability and transmission capability, and the existing novel crown vaccines all show different degrees of protective efficacy reduction. Therefore, the development of mRNA vaccines that are effective against SARS-CoV-2 wild strains and various mutant strains is an urgent need to suppress epidemic situations.
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, and (5) producing higher levels of neutralizing antibodies.
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 as 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 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 SEQ ID No.5 or SEQ ID No. 6.
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 set forth in SEQ ID No.1 or SEQ ID No. 2. In certain embodiments, the nucleotide sequence of the RNA is set forth 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%, 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.1 or SEQ ID No. 2.
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. 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%, at least 98%, or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID No. 9.
In certain embodiments, the RNA comprises a nucleotide sequence as 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%, 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.10 or SEQ ID No. 11.
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. 5-6.
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 SEQ ID No.7 or SEQ ID No. 8. In certain embodiments, the nucleotide sequence of the DNA is shown as SEQ ID NO.7 or SEQ ID NO. 8. 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 SEQ ID No.7 or SEQ ID No. 8.
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 SEQ ID No.3 or SEQ ID No. 4. In certain embodiments, the nucleotide sequence of the DNA is set forth in SEQ ID No.3 or SEQ ID No. 4. 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 SEQ ID No.3 or SEQ ID No. 4.
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%.
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, both mRNAs provided by the application can be translated in cells to generate high-level novel coronavirus Delta strain 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 two mRNAs provided by the invention are CVG024 and CVG025. Both mRNAs comprise open reading frames for encoding the whole length of the S protein of the novel coronavirus, the open reading frames of both mRNAs are subjected to codon optimization, the CVG024 whole length sequence is shown as SEQ ID NO.3, the CVG025 whole length sequence is shown as SEQ ID NO.4, and the encoded protein sequences are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6. There are mutations at multiple sites of the Delta strain S protein compared to the Wuhan-Hu-1 strain S protein. The protein sequences SEQ ID NO.5 and SEQ ID NO.6 expressed by the mRNA of the present invention each contain a plurality of mutations which occur in the S protein of the novel coronavirus Delta strain, such as T19R, T95I, G142D, E G, DEL/158, L452R, T478K, D614G, P681R, D950N; the protein sequence SEQ ID NO.6 also contains the K417N mutation found in the Delta strain S protein. Both 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 both mRNA sequences were all replaced by m1 ψ (1-methyl-3' -pseudouridyyl).
Drawings
FIG. 1 shows the results of mRNA expression in HEK 293T/17 cells as described in example 2.
FIG. 2 is a Western Blot detection of translation products following transfection of HEK 293T/17 cells in example 2.
FIG. 3 shows particle size and uniformity of mRNA-LNP coated with CVG025 mRNA of example 3 at Room Temperature (RT) or after thawing at-80 ℃.
FIG. 4 shows the encapsulation efficiency of mRNA-LNP coated with CVG025 mRNA in example 3 at Room Temperature (RT) or after thawing at-80 ℃.
FIG. 5 is a surface potential profile of mRNA-LNP coated with CVG025 mRNA of example 3 at Room Temperature (RT) or after thawing at-80 ℃.
FIG. 6 is a graph showing neutralizing antibody titers against Delta pseudovirus in mouse serum one week after the initial immunization in example 4; the abscissa corresponds from left to right to Saline, CVG025 1 ug/min, CVG025 10 ug/min, CVG025 ug/min.
FIG. 7 is a plot of neutralizing antibody titer against Delta pseudovirus in mouse serum after two immunizations for one week in example 4, where WT refers to the SARS-CoV-2 wild-type strain; the abscissa corresponds from left to right to Saline, CVG025 1. Mu.g/CVG 025. Mu.g/CVG 10. Mu.g/CVG 25. Mu.g/CVG.
FIG. 8 is a graph showing the titer of neutralizing antibodies against Delta pseudovirus in mouse serum after two weeks of immunization in example 4, where WT refers to the SARS-CoV-2 wild-type strain; the abscissa corresponds from left to right to Saline, CVG025 1. Mu.g/CVG 025. Mu.g/CVG 10. Mu.g/CVG 25. Mu.g/CVG.
FIG. 9 shows that CVG 025-induced neutralizing antibodies cross-react with the Omicron BA.2 subtype.
FIG. 10 is a graph showing that CVG025 causes 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 + The number of T cells was increased, and the IL2+ lymphocytes were significantly increased in delta S protein stimulated mouse spleen cells (fig. 10I), while IL-5 was not elevated (fig. 10J).
FIG. 11 shows serological evaluation and T-cell immunization of CVG025 boosted mice after two SARS-Covid-2 inactivated vaccinations, wherein A: schematic of mRNA vaccine boosting protocol, B: neutralizing antibody titers were determined using a pseudovirus neutralization assay in mice boosted with CVG025, C-H: spleen cells were collected 4 weeks after secondary immunization,and stimulated with S protein of different variants for 48h, followed by CVG025 stimulation and flow cytometry for determination of CD69 in CD4 and CD 8T cells + T cell ratio, I-J: ratio of Tfh (Foxp 3-pd1+) to DC (cd11c+ia/ie+) in spleen.
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.1-11" has the meanings given 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 the agents, components or pharmaceutically formulated compositions of the present application. 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 shown in SEQ ID No.5 or SEQ ID No. 6.
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 according to any one of embodiments 1-3 comprising a nucleotide sequence as set forth 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: 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. 9.
9. The RNA according to any one of embodiments 1-8 comprising a nucleotide sequence as set forth 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 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, comprising the nucleotide sequence shown as 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 to 14, which comprises the nucleotide sequence shown as 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 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 entrusting company synthesizes two 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.7 and SEQ ID NO.8, and the translated protein sequences are respectively shown as SEQ ID NO.5 and SEQ ID NO.6. Preferably, both genes are synthesized with a 5'UTR DNA sequence, a 3' UTR DNA sequence, a poly (A) tail. The synthetic gene product (sequence shown as SEQ ID NO.3 and SEQ ID NO. 4) comprising the 5'UTR, open reading frame, 3' UTR, poly (A) tail was cloned into the pUC57 vector. 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 T7 RNA 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. Two mRNAs were prepared and named CVG024 and CVG025, and the nucleic acid sequences were shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.
Example 2 mRNA cell translatability assay
CVG025mRNA was prepared according to the procedure described in example 1, and CVG025mRNA was mixed with transfection reagents in the indicated proportions, and transfected into HEK 293T/17 cells, and expression of the mRNA after transfection of the cells was detected, as shown in FIG. 1, and in FIG. 1, expression of the mRNA in HEK 293T/17 cells was shown. 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 new coronavirus S protein molecular weight size, neg ctrl.#1 and Neg ctrl.#2 are negative controls, and no expression of the new coronavirus S protein was observed; positive ctrl.#1 and Positive ctrl.#2 are Positive controls, and expression of novel coronavirus S protein was observed; CVG025mRNA #1- #4 is a four-group sample prepared independently, each of which expressed a protein product of the same size as the molecular weight of the novel coronavirus S protein. The results of fig. 1 and 2 show that: CVG025mRNA can be translated into S protein of the novel coronavirus.
EXAMPLE 3 Liposome nanoparticle 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 CVG025 mRNA at Room Temperature (RT) and after freeze thawing at-80 ℃ are shown in figure 3, the LNP diameter in both cases is between 80 and 100nm, the dispersion degree of the LNP particle diameter is measured by PDI, and the PDI values are lower, which indicates that the mRNA-LNP size is more uniform. The encapsulation efficiency of mRNA almost reaches 100% after freeze thawing of mRNA-LNP coated with CVG025 mRNA at Room Temperature (RT) and-80 ℃, as shown in FIG. 4, which shows that the novel mRNA corona 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 CVG025 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 CVG025 mRNA 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 1. Mu.g/dose, 10. Mu.g/dose, and 25. Mu.g/dose, respectively. Seven days after the first immunization, mice were collected and tested for neutralizing antibody titers against Delta pseudovirus or wild-type novel coronavirus in serum, 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 CVG025 mRNA and liposome nano particles prepared from liposome components are injected into a mouse body to induce and generate high-level neutralizing antibodies aiming at wild type and mutant new coronavirus strains, the level of the neutralizing antibodies is increased along with the increase of the concentration of the liposome nano particles, and the titer of a Delta mutant strain group is higher.
CVG024mRNA with the nucleic acid sequence shown in SEQ ID NO.1 has similar immune effect as CVG025mRNA, both mRNAs can be translated in cells to generate high-level novel coronavirus S protein, and each mRNA is injected into mice through a preparation formed by encapsulating liposome nano particles to induce the mice to generate high-titer neutralizing antibodies. The two 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 025-induced Cross-reactivity of neutralizing antibodies with Omicron subspecies
Mice were immunized twice with CVG025 on days 0 and 14, and serum samples from animals immunized for the second 14 days were incubated with Omacron BA.1 and BA.2 pseudoviruses and tested for neutralization activity. The results (fig. 9) show that CVG 025-induced antibodies cross-react with omacron ba.1 and ba.2, and ba.1 and ba.2 levels are similar.
EXAMPLE 6 CVG025 Induction of cellular immunity
This example examined T cell immune responses in CVG025 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 CVG025 can elicit antigen-specific responses in CD8 and CD 4T cells 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 by CVG025 produced a strong immune response to the new coronavirus. ELSA detection of IL2 expression in spleen cells from S protein stimulated mice also showed a significant increase in IL2+ lymphocytes from CVG025 vaccinated mice (FIG. 10I). At the same time, IL-5 was not elevated in all samples (FIG. 10J), indicating that CVG025 mainly induced a specific Th1 immune response, rather than Th2.
EXAMPLE 7 CVG025 boosting immunization
Balb/C mice were first immunized with BBIBP-CorV vaccine (2 doses, beijing Biotechnology institute of pharmaceutical group, china) prepared from inactivated wild virus, and then vaccinated with 3 rd CVG025 booster needle. Similarly, balb/C mice in the control group were vaccinated with a 3 rd needle BIBP-CorV homology boosting needle.
As shown in FIG. 11, after two doses of BBIBP-CorV immunization, one dose of CVG025 was inoculated to induce neutralizing antibodies, and antibodies were produced in each of the three strains (WT, delta, omicron), increasing CD69 + T cell ratio, T cell immunity is enhanced.
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 (15)
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.5 or SEQ ID No. 6.
2. The RNA of claim 1, comprising a nucleotide sequence as set forth in SEQ ID No.1 or SEQ ID No. 2.
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. 9.
5. The RNA of any one of claims 1-4, comprising a nucleotide sequence as set forth 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 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.7 or SEQ ID No. 8.
9. The DNA of claim 8, wherein said DNA further comprises the structure of the group consisting of: a 5 'untranslated sequence, a 3' untranslated sequence, and a poly (A) tail.
10. The DNA of any one of claims 8-9, comprising the nucleotide sequence set forth in SEQ ID No.3 or SEQ ID No. 4.
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.
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