WO2023169506A1 - 编码新型冠状病毒S蛋白的mRNA疫苗 - Google Patents

编码新型冠状病毒S蛋白的mRNA疫苗 Download PDF

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WO2023169506A1
WO2023169506A1 PCT/CN2023/080449 CN2023080449W WO2023169506A1 WO 2023169506 A1 WO2023169506 A1 WO 2023169506A1 CN 2023080449 W CN2023080449 W CN 2023080449W WO 2023169506 A1 WO2023169506 A1 WO 2023169506A1
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rna
seq
dna
mrna
protein
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French (fr)
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贾威廉
刘晓虎
陆昌瑞
杨晓明
张云涛
侯富军
杨国环
程新华
余志斌
丁隽
林康
徐江
华先武
潘欣萍
刘连晓
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中生复诺健生物科技(上海)有限公司
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Definitions

  • 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.
  • 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.
  • 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%. The new coronavirus (SARS-CoV-2) has caused a global pandemic.
  • recombinant protein vaccines require 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).
  • ADE antibody-dependent enhancement
  • 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
  • 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.
  • 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, (5) producing higher levels of neutralizing antibodies, and/or (6) producing an immune response that is protective against wild strains and multiple mutants of the new coronavirus.
  • the application provides an RNA 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. 7-8.
  • the amino acid sequence of the novel coronavirus S protein is as shown in any one of SEQ ID NO. 7-8.
  • 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.
  • the RNA is mRNA.
  • the nucleotide sequence of the RNA is codon optimized.
  • the RNA comprises the nucleotide sequence shown in any one of SEQ ID NOs. 13-15. In certain embodiments, the nucleotide sequence of the RNA is as shown in any one of SEQ ID NO. 13-15. In certain embodiments, the sequence of the RNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 96% similarity with the nucleotide sequence shown in any one of SEQ ID NO. 13-15. %, at least 97%, at least 98%, or at least 99% sequence identity.
  • 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.
  • the poly(A) tail of the RNA contains more than 30 adenosines.
  • the structure of the poly(A) tail is: 30 adenylate, a connecting sequence and 70 adenylate.
  • the poly(A) tail may be 100-200 nucleotides in length, such as about 120 nucleotides in length. For example, about 110 nucleotides.
  • the linker sequence can be 5-20 nucleotides in length. In certain embodiments, the linking sequence is GCAUAUGACU.
  • the poly(A) tail of the RNA comprises the nucleotide sequence set forth in SEQ ID NO. 12. In certain embodiments, the poly(A) tail has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% similarity with the nucleotide sequence shown in SEQ ID NO.12. %, at least 98%, or at least 99% sequence identity.
  • the RNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs. 1-3.
  • 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 any one of SEQ ID NO.1-3. %, at least 97%, at least 98%, or at least 99% sequence identity.
  • the RNA includes at least one modified nucleotide.
  • 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.
  • 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.
  • 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.
  • the chemical structural formulas of the first and second structural units in the 5'Cap structure are as follows:
  • 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).
  • 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. 7-8.
  • the present application provides a DNA transcribing the RNA described in the present application.
  • the DNA is codon optimized.
  • the DNA comprises the nucleotide sequence shown in any one of SEQ ID NOs. 9-11.
  • the nucleotide sequence of the DNA is as shown in any one of SEQ ID NO. 9-11.
  • 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 any one of SEQ ID NO. 9-11. %, at least 97%, at least 98%, or at least 99% sequence identity.
  • the DNA further includes a 5' untranslated sequence, a 3' untranslated sequence, and a poly(A) tail.
  • the DNA comprises the nucleotide sequence shown in any one of SEQ ID NOs. 4-6. In certain embodiments, the nucleotide sequence of the DNA is as shown in any one of SEQ ID NO. 4-6. In certain 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 any one of SEQ ID NO. 4-6. %, at least 97%, at least 98%, or at least 99% sequence identity.
  • 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.
  • the mRNA can be encapsulated within nanoparticles.
  • the nanoparticles include lipids.
  • Lipid nanoparticles may include, but are not limited to, liposomes and micelles.
  • 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.
  • lipid nanoparticles comprise one or more RNAs described herein, such as mRNA, and for example, mRNA encoding S protein.
  • 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.
  • KL10 3-(didodecylamino)-N1,
  • 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%.
  • 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
  • the molar proportion of the phospholipids in the lipid nanoparticles is about 5-20%.
  • 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.
  • the polyethylene glycol (PEG) modified lipid molecule can be a PEG-ceramide with a shorter acyl chain (eg, C14 or C18).
  • 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%.
  • the The polyethylene glycol (PEG) modified lipid molecule can be PEG2000-DMG.
  • the nanolipid particles may also contain cholesterol.
  • the molar proportion of cholesterol in the lipid nanoparticle is about 30-50%, for example, about 35-45%, or about 38-42%.
  • the nanolipid particles may include cationic lipids, cholesterol, phospholipids, and polyethylene glycol-modified lipid molecules.
  • 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.
  • the application provides a composition comprising the RNA, and a delivery vehicle.
  • the present application provides a composition, which may include the mRNA described in the present application and may also include a delivery carrier.
  • the delivery vehicle includes liposomes.
  • the delivery vehicle includes lipid nanoparticles (LNP).
  • LNP lipid nanoparticles
  • the lipid nanoparticles include cationic lipids, non-cationic lipids, and cholesterol.
  • the present application provides a lipid nanoparticle formulation that coats the RNA.
  • the present application provides a vaccine comprising the RNA, the DNA, the composition, and/or the lipid nanoparticle formulation.
  • the vaccine includes an RNA vaccine, a DNA vaccine, a recombinant vaccine, and/or an adenovirus vaccine.
  • 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.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the RNA, the DNA, the composition, the liposome nanoparticle formulation, the vaccine, and/or a pharmaceutically acceptable composition thereof Carrier.
  • 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.
  • 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.
  • the in vivo nanoparticle formulation, the pharmaceutical composition and/or the vaccine can be administered in a single, multiple or continuous manner and are safe. After the initial administration, subsequent administration of the composition and/or pharmaceutical composition may shorten the onset of action.
  • 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.
  • 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.
  • 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.
  • the disease or condition associated with novel coronavirus infection includes pneumonia, such as COVID-19.
  • the drugs eg. the vaccines
  • the drugs can be administered sequentially.
  • the drug eg, the vaccine
  • the drug can be administered multiple times, eg, two, three, four, or more times.
  • the drug eg, the vaccine
  • 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.
  • the medicine of the present application for example, the vaccine
  • the medicine of the present application 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).
  • 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.
  • the present application provides a vector, which includes the RNA and the DNA.
  • the vector may be a viral vector, such as an adenoviral vector, an adeno-associated viral vector, and/or a lentiviral vector.
  • a viral vector such as an adenoviral vector, an adeno-associated viral vector, and/or a lentiviral vector.
  • the application provides cells comprising said nucleic acid molecule, and/or said vector.
  • the cell may be a prokaryotic cell, for example, E. coli.
  • the cells may be eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells.
  • the cells may be mammalian cells, such as mouse cells, human cells, etc.
  • the vector comprises a viral vector.
  • the present application provides a kit comprising the RNA, the DNA, the composition, the liposomal nanoparticle formulation, the vaccine, and/or the pharmaceutical composition.
  • 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.
  • 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.
  • the method is an in vitro or ex vivo method.
  • the three types of mRNA provided by the present invention can be translated in cells and produce high levels of the new coronavirus S protein. Each mRNA is injected into mice through a preparation formed after encapsulating liposome nanoparticles to induce mice. 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.
  • the three types of mRNA provided by the present invention are CVG031, CVG032 and CVG033.
  • the three mRNAs all contain open reading frames encoding the full length of the new coronavirus S protein.
  • the open reading frames of the three mRNAs have been codon-optimized.
  • the full-length sequence of CVG031 is as SEQ ID NO.1
  • the full-length sequence of CVG032 is as SEQ ID NO.
  • the full-length sequence of CVG033 is shown in SEQ ID NO.3
  • CVG031 and CVG033 encode the same protein sequence, using different codon optimization strategies, and the encoded protein sequence is shown in SEQ ID NO.7; CVG032
  • the encoded protein sequence is shown in SEQ ID NO. 8.
  • the S protein of the Omicron strain has mutations at multiple sites.
  • the protein sequences SEQ ID NO.7 and SEQ ID NO.8 expressed by the mRNA of the present invention both contain multiple mutations that appear in the S protein of the new coronavirus Omicron strain, such as A67V, ⁇ 69-70, T95I, G142D, ⁇ 143-145 , ⁇ 211,L212I,ins214EPE,G339D,S371L,S373P,S375F,K417N,N440K,G446S,S477N,T478K,E484A,Q493R,G496S,Q498R,N501Y,Y505H,T547K,D614G, H655Y,N679K,P681H,N764K,D796Y , N856K, Q954H, N969K, L981F; the protein sequence SEQ ID NO.7 and SEQ ID NO.8 expressed by
  • the above SEQ ID NO.7 protein sequence contains the S-2P mutation, which It can stabilize the S protein conformation in the pre-fusion state, and the stable pre-fusion S protein S-2P mutation has better immunogenicity than the wild-type S protein.
  • the three kinds of mRNA all contain 5'Cap structure, 5'untranslated sequence (5'UTR), 3'untranslated sequence (3'UTR) and poly(A) tail.
  • the structural unit U bases in the three mRNA sequences are all replaced by m1 ⁇ (1-methyl-3'-pseudouridylyl).
  • Figure 1 shows the expression results of mRNA in HEK 293T/17 cells in Example 2 of the present invention.
  • Figure 2 shows Western Blot detection of translation products after mRNA transfection into HEK 293T/17 cells in Example 2 of the present invention.
  • Figure 3 shows the particle size and uniformity of the mRNA-LNP coated with CVG031 mRNA in Example 3 of the present invention at room temperature (RT) and after freezing and thawing at -80°C.
  • Figure 4 shows the encapsulation efficiency of CVG031 mRNA-coated mRNA-LNP at room temperature (RT) and after freezing and thawing at -80°C in Example 3 of the present invention.
  • Figure 5 shows the surface potential properties of the mRNA-LNP coated with CVG031 mRNA in Example 3 of the present invention at room temperature (RT) and after freezing and thawing at -80°C.
  • Figure 6 shows the neutralizing antibody titer against Omicron pseudovirus in mouse serum one week after the initial immunization in Example 4 of the present invention; the abscissas from left to right correspond to Saline, CVG031 3 ⁇ g/mouse, and CVG031 10 ⁇ g/mouse. , CVG031 30 ⁇ g/piece.
  • Figure 7 shows the neutralizing antibody titers against wild strain pseudovirus, Delta pseudovirus, and Omicron pseudovirus in mouse serum one week after two immunizations in Example 4 of the present invention, where WT refers to SARS-CoV- 2 wild strains; the abscissas from left to right correspond to Saline, CVG031 3 ⁇ g/bird, CVG031 10 ⁇ g/bird, and CVG031 30 ⁇ g/bird.
  • Figure 8 shows the neutralizing antibody titers against wild strain pseudovirus, Delta pseudovirus, and Omicron pseudovirus in mouse serum two weeks after two immunizations in Example 4 of the present invention, where WT refers to SARS-CoV. -2 wild strains; the abscissas from left to right correspond to Saline, CVG031 3 ⁇ g/bird, CVG031 10 ⁇ g/bird, and CVG031 30 ⁇ g/bird.
  • Figure 9 shows that neutralizing antibodies induced by CVG031 can cross-react with Omicron subtypes.
  • Figure 10 shows the antigen-specific responses caused by CVG031 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 IFN ⁇ + /CD69 + T cells increased after treatment with Spike protein.
  • Figure 11 shows the protective effect of CVG031 heterologous boosting in mice against SARS-Cov-2 Delta and Omicron variant challenge.
  • A Experimental design of Delta and Omicron variant challenge protection test
  • B and C After virus infection Survival rate of each group
  • D and E body weight changes of animals after infection.
  • 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.
  • 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.
  • 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.
  • DNA generally includes cDNA or genomic DNA
  • 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.
  • mRNA generally refers to an RNA transcript that has been processed to remove introns and capable of being translated into a polypeptide.
  • the term "modification" when applied to a nucleic acid 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.
  • the modification may include modification of a nucleotide, for example, the nucleotide may include a modified base, sugar, or phosphate group.
  • 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 or introduce side groups not naturally found in these nucleic acid molecules).
  • 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.
  • modified nucleic acids such as modified mRNA
  • 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-amino
  • RNA molecules 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%.
  • the RNA molecule includes about 0.1% modified nucleotides to about 100% modified nucleotides.
  • 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.
  • 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.
  • 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.
  • active ingredient eg, vaccine, composition, pharmaceutical composition, vector, cell, DNA or RNA of the present application
  • these preparations can be sterile.
  • 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.
  • vaccine generally refers to a treatment containing a substance that is effective in inducing resistance in a subject to a specific pathogen or disease.
  • An agent or composition containing a therapeutically potent immunologically active ingredient is an agent or composition containing a therapeutically potent immunologically active ingredient.
  • 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).
  • 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.
  • 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.
  • 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).
  • pharmaceutically acceptable carrier generally refers to any solvent, dispersion medium, coating, isotonic and absorption delaying agents and other adjuvants, excipients or Other drug carriers.
  • 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.
  • the term "effective amount” refers to an amount capable of treating or ameliorating a disease or condition or producing the desired therapeutic effect.
  • the term “homology” or “identity” refers to the degree of complementarity between two or more polynucleotide or polypeptide sequences.
  • identity refers to the degree of complementarity between two or more polynucleotide or polypeptide sequences.
  • 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.
  • 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 Examine measurements to compare and align for maximum correspondence when two or more sequences or subsequences are identical or have a specific percentage of similarity. identical amino acid residues or nucleotides.
  • 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.
  • kits 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.
  • such kits may include one or more sets of instructions for use of the encapsulated compositions, for example, in laboratory or clinical applications.
  • 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.
  • RNA 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. 7-8.
  • RNA according to embodiment 1 which is mRNA.
  • RNA of any one of embodiments 1-2 whose nucleotide sequence is codon-optimized.
  • RNA of any one of embodiments 1-3 which comprises the nucleotide sequence shown in any one of SEQ ID NO. 13-15.
  • 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.
  • RNA of embodiment 5 wherein the poly(A) tail contains more than 30 adenosines.
  • RNA of any one of embodiments 5-7, wherein the poly(A) tail comprises the nucleotide sequence shown in SEQ ID NO. 12.
  • RNA of any one of embodiments 1-8 which comprises the nucleotide sequence shown in any one of SEQ ID NO. 1-3.
  • RNA of any one of embodiments 1-9 comprising at least one modified nucleotide.
  • 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.
  • 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).
  • N1-methylpseudouridine triphosphate N1-methylpseudouridine triphosphate
  • pseudo-UTP pseudouridine triphosphate
  • 5-methoxyuridine triphosphate 5-Methoxy-UDP
  • 5-methylcytidine triphosphate 5-Methyl-CTP
  • composition comprising the RNA of any one of embodiments 1-12, and a delivery vehicle.
  • composition of embodiment 16, wherein the delivery vehicle comprises liposomes.
  • LNP lipid nanoparticles
  • 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 sodium according to embodiment 20 Rice granule preparation.
  • 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
  • a method for treating and/or preventing diseases or conditions related to novel coronavirus infection 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.
  • 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.
  • a vector comprising the RNA described in any one of embodiments 1-12 and the DNA described in any one of embodiments 13-15.
  • the vector of embodiment 25, comprising a viral vector.
  • 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 .
  • kits 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.
  • 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.
  • 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 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.
  • RNA in vitro transcription 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 are as follows:
  • the company was commissioned to synthesize three 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.9, SEQ ID NO.10, and SEQ ID NO.11.
  • SEQ ID NO.9 The protein sequence translated from SEQ ID NO. 10 is the same as SEQ ID NO. 7, and the protein sequence translated from SEQ ID NO. 11 is shown as SEQ ID NO. 8.
  • the three genes are synthesized together with the 5'UTR DNA sequence, the 3'UTR DNA sequence, and the poly(A) tail.
  • the synthetic gene product containing 5'UTR, open reading frame, 3'UTR, and poly(A) tail was cloned into the pUC57 vector. After the vector is amplified, it is digested with restriction endonuclease to obtain the linearized vector. The purified linearized vector was used as a template for RNA in vitro transcription, and T7 RNA polymerase, CTP, GTP, ATP, and m1 ⁇ (1-methyl-3'-pseudouridylyl)-modified UTP (i.e., N1-methylpseudouridine trisulfide) were added.
  • Phosphate N1-Methylpseudo-UTP
  • Cap analog ((3'-OMe-m7G)(5')ppp(5')(2'-OMeA)pG, that is, the 3' hydroxyl group of m7G is methylated Modification, ensuring higher 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. Three kinds of mRNA were prepared, named CVG031, CVG032, and CVG033, and their nucleic acid sequences are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively.
  • Spike in Figure 2 indicates the molecular weight of the new coronavirus S protein
  • Ctrl is the negative control group, and no expression of the new coronavirus S protein was observed; the LNP/CVG031 group containing CVG031 mRNA expressed a protein product with the same molecular weight of the new coronavirus S protein.
  • the results in Figures 1 and 2 show that CVG031 mRNA can be translated into the S protein of the new coronavirus.
  • Example 1 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-component lipid mixture of Example 1 were made into mRNA-LNPs using a microfluidic nanoprecipitation process in which an aqueous solution of mRNA at acidic pH was rapidly mixed with an ethanolic solution of lipids.
  • MC3 cationic lipid
  • DSPC DSPC
  • cholesterol and PEG-lipid dissolved in ethanol at a molar ratio of 50:10:38.5:1.5.
  • Lipid nanoparticles are prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. Briefly, the mRNA and four-component
  • 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 ⁇ m Sartopore PES membrane, and aliquots are subsequently stored at room temperature or frozen at -80°C (1.0 mL fill).
  • mRNA-LNP 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.
  • EE encapsulation efficiency
  • 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 size of mRNA-LNP coated with CVG031 mRNA at room temperature (RT) and after freezing and thawing at -80°C The uniformity is shown in Figure 3.
  • the LNP diameter in the two cases is almost unchanged, both between 60-80nm, indicating that CVG031 mRNA-LNP is very stable.
  • the mRNA encapsulation rate of the mRNA-LNP coated with CVG031 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 CVG031 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 3 After filtering the liposome nanoparticle preparation coated with CVG031 mRNA in Example 3, it was injected into 6-8 week female Balb/c mice. The control group was injected with physiological saline (Saline). The injection dose of the liposome nanoparticle preparation was 3 ⁇ g respectively. /piece, 10 ⁇ g/piece, 30 ⁇ g/piece. Seven days after the first immunization, the blood of the mice was collected and the neutralizing antibody titer against the Omicron new coronavirus pseudovirus in the serum was detected. The results are shown in Figure 6. On the 21st day after the primary immunization, the mice were injected again with the filtered liposome nanoparticle preparation of Example 3. This was the secondary immunization.
  • Saline physiological saline
  • the nucleic acid sequences such as CVG032mRNA and CVG033mRNA shown in SEQ ID NO.2 and SEQ ID NO.3 have similar immune effects to CVG031mRNA. All three mRNAs can be translated in cells and produce high levels of the new coronavirus S protein. Each mRNA was injected into mice via a preparation formed after encapsulation with liposome nanoparticles, inducing the mice to produce high-titer neutralizing antibodies. Compared with other mRNAs or mRNAs without codon optimization, the three mRNAs of this application can stimulate stronger immune responses and produce higher levels of neutralizing antibodies.
  • mice were immunized twice with CVG031 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. Results (Fig. 9) showed that antibodies induced by CVG031 cross-reacted with Omicron BA.1 and BA.2.
  • Example 6 CVG031 induces cellular immunity
  • This example detects the T cell immune response of animals immunized with CVG031. 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 CVG031 can induce antigen-specific responses of CD8 and CD4 T cells, which can be seen from the increase in the number of IFN ⁇ + /CD69 + T cells after treatment with wild-type, Delta or Omicron's Spike protein, respectively (Fig. 10A- Figure 10H).
  • mice immunized with CVG031 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 CVG031 ( Figure 10I).
  • IL-5 was not increased in all samples ( Figure 10J), indicating that CVG031 mainly induces a specific Th1 immune response rather than Th2.
  • FIG. 11A shows that 14 days after completion of 2 doses of BBIBP-CorV, animals received a booster injection of different doses of BBIBP-CorV or CVG031. Fourteen days after the booster injection, the animals were inoculated intranasally with 200 CCID50 of Delta or Omicron virus. In the saline control group, 4/4 of the animals died within 6 days of infection with the Delta variant.
  • BBIBP-CorV Beijing Institute of Biological Products of China National Biotech Group

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Abstract

提供了编码新型冠状病毒S蛋白的mRNA疫苗。所述mRNA编码的S蛋白包含如SEQ ID NO.7-8中任一项所示的氨基酸序列。所述mRNA包含SEQ ID NO.1-3中任一项所示的核苷酸序列。提供的三种mRNA都能在细胞内翻译，产生高水平的新型冠状病毒S蛋白，每种mRNA经由脂质体纳米颗粒包封后形成的制剂注射到小鼠体内，诱导小鼠产生高滴度中和抗体。

Description

编码新型冠状病毒S蛋白的mRNA疫苗 技术领域
本发明属于生物疫苗技术领域，具体涉及编码新型冠状病毒S蛋白的mRNA疫苗及其应用。
背景技术
疫苗是预防传染性疾病最有效的公共卫生干预手段。世界卫生组织估算，由于接种疫苗，每年避免了两百至三百万人死亡。尽管传统的疫苗类型已经在公共卫生事业方面发挥了巨大的作用，但人类仍然面临新型传染病及其变异株的威胁。开发针对新型传染病的疫苗，革新疫苗生产方式，提高疫苗的有效性，缩短疫苗的生产周期，是推动公共卫生事业进步的迫切需求。
冠状病毒是单股正链RNA病毒，由外层囊膜结构包裹基因组，具有传播能力强、容易变异等特点，能够感染多种动物，可在人类中引起严重急性呼吸综合征。进入二十一世纪，已经出现SARS-CoV、MERS-CoV和SARS-CoV-2三种高致病性冠状病毒，对人类健康带来严重威胁。SARS-CoV全球致死率达10％，MERS-CoV全球致死率达35％。新型冠状病毒(SARS-CoV-2)造成全球大流行，根据世界卫生组织(WHO)官网数据，截至2022年1月10日，全球COVID-19确诊病例已经超过3亿。针对新型冠状病毒开发疫苗是遏制新型冠状病毒大流行的迫切需求。
目前，多条不同的疫苗开发路线同时进行，如重组蛋白疫苗、灭活疫苗、载体疫苗。重组蛋白疫苗的生产需要大规模体外细胞的培养，并从培养物中分离纯化抗原蛋白，对蛋白表达和纯化的工艺要求较高；灭活疫苗需要筛选合适的毒株，耗时长，培养活病毒具有一定风险，灭活病毒的工艺要求较高。灭活的病毒亦有返祖和抗体依赖增强作用(ADE)的风险。
mRNA疫苗具有很高的安全性和延展性，并且易于提高产量，因此受到科研人员和医药机构的关注，mRNA在疫苗开发和生产上的应用具有广阔前景。
S蛋白(spike)是新型冠状病毒重要的表面蛋白。S蛋白上的RBD结构域结合人体细胞表面的ACE2蛋白，从而介导SARS-CoV-2病毒进入细胞。S蛋白是宿主体内中和抗体的主要作用位点。假病毒中和试验表明，针对S蛋白的中和抗体降低了病毒侵染细胞的 阳性率。因此，S蛋白是新型冠状病毒疫苗开发最主要的抗原蛋白。Moderna和BioNtech公司已经上市的mRNA疫苗均编码S蛋白全长，并在大规模推广接种中显示出极高的保护效力和安全性。但是，随着在人群中感染的时间增长，已检测到了SARS-CoV-2的多种突变毒株，例如Alpha毒株、Beta毒株、Gamma毒株、Delta毒株、Omicron毒株。相比于野生型毒株，这些突变毒株具有更强的侵染能力和传播能力，现有新冠疫苗均显示出了不同程度的保护效力下降。因此，开发有效针对SARS-CoV-2野生毒株和多种突变毒株的mRNA疫苗是遏制疫情的迫切需求。
发明内容
本申请提供了一种编码新型冠状病毒S蛋白的RNA，以及包含所述RNA的疫苗。本申请的mRNA或疫苗具有以下一种或多种特点：(1)更高的蛋白表达量，(2)能够刺激更强的免疫反应，(3)产生更多的细胞因子，(4)具有降低的免疫原性，(5)产生更高水平的中和抗体，和/或(6)产生的免疫应答对新冠病毒野生株和多种突变体都有保护效力。
一方面，本申请提供了一种编码新型冠状病毒S蛋白的RNA，其中所述新型冠状病毒S蛋白包含SEQ ID NO.7-8中任一项所示的氨基酸序列。在某些实施方案中，所述新型冠状病毒S蛋白的氨基酸序列如SEQ ID NO.7-8中任一项所示。在某些实施方案中，所述S蛋白包含与SEQ ID NO.7-8中任一项所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列。
在某些实施方式中，所述RNA为mRNA。
在某些实施方式中，所述RNA的核苷酸序列是密码子优化的。
在某些实施方式中，所述RNA包含SEQ ID NO.13-15中任一项所示的核苷酸序列。在某些实施方式中，所述RNA的核苷酸序列如SEQ ID NO.13-15中任一项所示。在某些实施方式中，所述RNA的序列与SEQ ID NO.13-15中任一项所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在某些实施方式中，所述RNA包含选自下组的一种或多种结构：5'Cap结构、5'非翻译序列、开放阅读框、3'非翻译序列和poly(A)尾。
在某些实施方式中，所述RNA的所述poly(A)尾包含超过30个腺苷酸。
在某些实施方式中，所述RNA中，自5’端至3’端，所述poly(A)尾的结构为：30个腺苷酸、连接序列和70个腺苷酸。例如，所述poly(A)尾的长度可以为100-200个核苷酸，例如约120个核苷酸。例如约110个核苷酸。
在某些实施方式中，所述连接序列的长度可以为5-20个核苷酸。在某些实施方式中，所述连接序列为GCAUAUGACU。
在某些实施方式中，所述RNA的所述poly(A)尾包含SEQ ID NO.12所示的核苷酸序列。在某些实施方式中，所述poly(A)尾与SEQ ID NO.12所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在某些实施方式中，所述RNA包含如SEQ ID NO.1-3中任一项所示的核苷酸序列。在某些实施方式中，所述RNA的序列与SEQ ID NO.1-3中任一项所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在某些实施方式中，所述RNA包含至少一种经修饰的核苷酸。
在某些实施方式中，所述RNA在选自下组的一个或多个位置处包含修饰：5'Cap结构、5'非翻译序列、开放阅读框、3'非翻译序列和poly(A)尾。例如，所述5'非翻译序列可包含选自下组的基因的5'非翻译序列或其同源物、片段或变体：β-珠蛋白基因、热休克蛋白70基因、轴丝动力蛋白重链2基因、羟基类固醇(17-β)脱氢酶基因，和/或KOZAK序列。例如，所述3'非翻译序列可包含选自下组的基因的3'非翻译序列或其同源物、片段或变体：白蛋白基因、α-珠蛋白基因、β-珠蛋白基因、酪氨酸羟化酶基因、热休克蛋白70基因，脂氧合酶基因和胶原蛋白α基因。
在某些实施方式中，所述5'Cap结构中第一和第二个结构单元的化学结构式如下：
在某些实施方式中，所述RNA的所述经修饰的核苷酸包含一种或多种包含选自下组的核苷酸：N1-甲基假尿苷三磷酸(N1-Methylpseudo-UTP)、假尿苷三磷酸(pseudo-UTP)、5-甲氧基尿苷三磷酸(5-Methoxy-UDP)和5-甲基胞苷三磷酸(5-Methyl-CTP)。
在某些实施方式中，所述RNA的序列中的结构单元U碱基全部被m1Ψ＝1-methyl-3'-pseudouridylyl替代。
另一方面，本申请提供了一种DNA，所述DNA编码新型冠状病毒S蛋白，其中所述新型冠状病毒S蛋白包含SEQ ID NO.7-8中任一项所示的氨基酸序列。
另一方面，本申请提供了一种转录本申请所述的RNA的DNA。
在某些实施方案中，所述DNA是密码子优化的。某些实施方案中，所述DNA包含SEQ ID NO.9-11中任一项所示的核苷酸序列。某些实施方案中，所述DNA的核苷酸序列如SEQ ID NO.9-11中任一项所示。在某些实施方式中，所述DNA的序列与SEQ ID NO.9-11中任一项所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在某些实施方式中，所述DNA还包括5'非翻译序列、3'非翻译序列和poly(A)尾。
在某些实施方式中，所述DNA包含SEQ ID NO.4-6中任一项所示的核苷酸序列。在某些实施方式中，所述DNA的核苷酸序列如SEQ ID NO.4-6中任一项所示。在某些实施方式中，所述DNA的序列与SEQ ID NO.4-6中任一项所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
本申请的RNA(或DNA)可以配制在纳米颗粒或其他递送载体中，例如，以避免它们在递送至受试者时被降解。在本申请中，所述mRNA可以被包封在纳米颗粒内。在特定的实施方案中，纳米颗粒包括脂质。脂质纳米颗粒可以包括但不限于脂质体和胶束。在本申请中，所述脂质纳米颗粒可以包括阳离子和/或可电离的脂质、阴离子脂质、中性脂质、两亲性脂质、聚乙二醇化的脂质和/或结构性脂质，或上述的组合在某些实施方案中，脂质纳米颗粒包含一种或多种本申请所述的RNA，例如mRNA，又例如，编码S蛋白的mRNA。
关于递送的具体方式，并无特别限制，可以采用本领域常规使用的那些。例如，可以参考已公开专利US20160376224A1或WO2015199952A1中所披露的方式。
本申请所述组合物中的递送载体可以是纳米脂质颗粒。所述纳米脂质颗粒可包含一种或多种(例如1、2、3、4、5、6、7或8)阳离子和/或可离子化的脂质。“阳离子脂质”通常指在一定pH(例如，生理pH)下携带任意数目的净正电荷的脂质。所述阳离子脂质可包括但不限于SM102、3-(双十二烷基氨基)-N1,N1,4-三十二烷基-1-哌嗪乙胺(KL10)、 N1-[2-(二十二烷基氨基)乙基]-N1,N4,N4-三十二烷基-1,4-哌嗪二乙胺(KL22)、14,25-二十三烷基-15,18,21,24-四氮杂八孔并烷(KL25)、DLin-DMA、DLin-K-DMA、DLin-KC2-DMA、Octyl-CLinDMA、辛基-CLinDMA(2S)、DODAC、DOTMA、DDAB、DOTAP、DOTAP.C1、DC-Choi、DOSPA、DOGS、DODAP、DODMA和DMRIE。
在某些实施方式中，所述阳离子脂质在脂质纳米颗粒中的摩尔比例为约40-70％，例如，约40-65％、约40-60％、约45-55％或约48-53％。
在本申请中，所述纳米脂质颗粒可包含一种或多种(例如1、2、3、4、5、6、7或8)非阳离子脂质。所述非阳离子脂质可以包括阴离子脂质。适用于本申请的脂质纳米颗粒的阴离子脂质可包括磷脂酰甘油、心磷脂、二酰基磷脂酰丝氨酸、二酰基磷脂酸、N-十二烷酰基磷脂酰乙醇胺、N-琥珀酰基磷脂酰乙醇胺、N-戊二酰基磷脂酰磷酸乙醇基，以及其他连接了阴离子基团的中性脂质。
所述非阳离子脂质可以包括中性脂质。适用于本申请的脂质纳米颗粒的中性脂质可包括磷脂，例如二硬脂酰基磷脂酰胆碱(DSPC)、二油酰基磷脂酰胆碱(DOPC)、二棕榈酰基磷脂酰胆碱(DPPC)、二油酰基磷脂酰甘油(DOPG)、二棕榈酰基磷脂酰甘油(DPPG)、二油酰基磷脂酰乙醇胺(DOPE)、棕榈酰基油酰基磷脂酰胆碱(POPC)、棕榈酰基油酰基-磷脂酰乙醇胺(POPE)、二油酰基-磷脂酰乙醇胺4-(N-马来酰亚胺基甲基)-环己烷-1-羧酸酯(DOPE-mal)、二棕榈酰基磷脂酰基乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、二硬脂酰基-磷脂酰基-乙醇胺(DSPE)、16-O-单甲基PE、16-O-二甲基PE、18-1-反式PE、1-硬脂酰基-2-油酰基-磷脂酰乙醇胺(SOPE)，或其混合物。另外，可以使用具有饱和和不饱和脂肪酸链的混合物的脂质。例如，本申请所述的中性脂质可以选自DOPE、DSPC、DPPC、POPC或任何相关的磷脂酰胆碱。
在某些实施方式中，所述磷脂在脂质纳米颗粒中的摩尔比例为约5-20％。
在本申请中，所述纳米脂质颗粒可包含脂质缀合物，例如，聚乙二醇(PEG)修饰的脂质和衍生的脂质。PEG修饰的脂质可包括但不限于与具有C6-C20长度的烷基链的脂质共价连接的长度至多为5kDa的聚乙二醇链。这些组分的加入可防止脂质聚集，也可增加循环持续时间，易于脂质-核酸组合物递送至靶细胞，或快速释放出核酸。例如，所述聚乙二醇(PEG)修饰的脂分子可以是具有较短的酰基链(例如，C14或C18)的PEG-神经酰胺。在某些实施方式中，所述聚乙二醇(PEG)修饰的脂分子在脂质纳米颗粒中的摩尔比例为约0.5-2％，例如，约1-2％、约1.2-1.8％或约1.4-1.6％。在某些实施方式中，所述 聚乙二醇(PEG)修饰的脂分子可以为PEG2000-DMG。
在本申请中，所述纳米脂质颗粒还可包含胆固醇。在某些实施方式中，所述胆固醇在脂质纳米颗粒中的摩尔比例为约30-50％，例如，约35-45％、或约38-42％。
在本申请中，所述纳米脂质颗粒可包括阳离子脂质、胆固醇、磷脂以及聚乙二醇修饰的脂分子。在某些实施方式中，所述阳离子脂质、胆固醇、磷脂以及聚乙二醇修饰的脂分子的摩尔比可以为45～55：35～45：5～15：0.5～2。
另一方面，本申请提供了一种组合物，所述组合物包含所述RNA，和递送载体。
另一方面，本申请提供了一种组合物，所述组合物可包含本申请所述的mRNA，还可以包含递送载体。
在某些实施方式中，所述递送载体包括脂质体。
在某些实施方式中，所述递送载体包括脂质纳米颗粒(LNP)。
在某些实施方式中，所述脂质纳米颗粒包括阳离子脂质、非阳离子脂质和胆固醇。
另一方面，本申请提供了一种脂质纳米颗粒制剂，其包被所述RNA。
另一方面，本申请提供了一种疫苗，其包括所述RNA、所述DNA、所述组合物，和/或所述脂质纳米颗粒制剂。在某些实施方式中，所述疫苗包含RNA疫苗、DNA疫苗、重组疫苗和/或腺病毒疫苗。
另一方面，本申请提供了一种制备RNA疫苗的方法，所述方法包括：a.将离子化脂质、聚乙二醇化脂质、胆固醇及衍生物和磷脂溶解在乙醇溶液中；b.通过微流控混合器将所述脂质乙醇溶液与RNA水溶液混合得到脂质纳米颗粒(LNP)；c.分离和纯化步骤b中获得的LNP从而获得所述RNA疫苗，其中所述RNA如本申请所定义。
另一方面，本申请提供了一种药物组合物，其包括所述RNA、所述DNA、所述组合物、所述脂质体纳米颗粒制剂、所述疫苗，和/或其药学上可接受的载体。
另一方面，本申请提供了一种治疗和/或预防新型冠状病毒感染相关的疾病或病症的方法，所述方法包括向有需要的患者施用所述RNA、所述DNA、所述组合物、所述脂质体纳米颗粒制剂、所述疫苗，和/或所述药物组合物。
在一些实施例中，本申请的药物组合物可以通过本领域技术人员已知的任何方法给予受试者，例如肠胃外、经口、经粘膜、经皮、肌肉内、静脉内、皮内、皮下、腹膜内、心室内、颅内、阴道内或肿瘤内。
在本申请中，所述RNA、所述DNA、所述载体、所述细胞、所述组合物、所述脂质 体纳米颗粒制剂、所述药物组合物和/或所述疫苗可以被单次施用、多次施用或连续施用，并且均具有安全性。在首次施用后，再次施用所述组合物和/或药物组合物可以缩短起效时间。在本申请中，所述RNA、所述DNA、所述载体、所述细胞、所述组合物、所述脂质体纳米颗粒制剂、所述药物组合物和/或所述疫苗可以和其他活性物质或治疗/预防成分共同施用。在本申请中，所述RNA、所述DNA、所述载体、所述细胞、所述组合物、所述脂质体纳米颗粒制剂、所述药物组合物和/或所述疫苗可以在其他活性物质或治疗/预防成分之前或之后施用。
另一方面，本申请提供了所述RNA、所述DNA、所述组合物、所述脂质体纳米颗粒制剂、所述疫苗，和/或所述药物组合物在制备药物中的用途，所述药物用于治疗和/或预防新型冠状病毒感染相关的疾病或病症。
在某些实施方式中，所述新型冠状病毒感染相关的疾病或病症包括肺炎，例如COVID-19。
在某些实施方式中，所述药物(例如，所述疫苗)可以序贯使用。
在某些实施方式中，所述药物(例如，所述疫苗)可以重复多次施用，例如，二次、三次、四次或以上。
在某些实施方式中，所述药物(例如，所述疫苗)可以与针对新型冠状病毒的其他疫苗进行前后序贯接种。针对新型冠状病毒的其他疫苗可以为现有技术中预防新型冠状病毒的其他任意疫苗，包括但不限于灭活疫苗、mRNA疫苗、DNA疫苗、重组蛋白疫苗和/或腺病毒疫苗。例如，可以先施用本申请的药物(例如，所述疫苗)，再施用针对新型冠状病毒的其他疫苗，也可以先施用针对新型冠状病毒的其他疫苗，再施用本申请的药物(例如，所述疫苗)。
在某些实施方式中，所述药物(例如，所述疫苗)序贯施用时，相邻两次施用的时间间隔可以为1天、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、1个月、2个月、3个月、4个月、5个月、6个月或更久。具体时间可以根据受试者体质、健康状况、施用首次疫苗后的免疫应答水平等确定。
另一方面，本申请提供了一种载体，其包括所述RNA、所述DNA。
例如，所述载体可以为病毒载体，例如，腺病毒载体、腺相关病毒载体和/或慢病毒载体。
另一方面，本申请提供了细胞，所述细胞包含所述核酸分子，和/或所述载体。在本 申请中，所述细胞可以是原核细胞，例如，大肠杆菌。在本申请中，所述细胞可以是真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞。在本申请中，所述细胞可以为哺乳动物的细胞，例如小鼠细胞、人细胞等。
在某些实施方式中，所述载体包含病毒载体。
另一方面，本申请提供了一种试剂盒，其包括所述RNA、所述DNA、所述组合物、所述脂质体纳米颗粒制剂、所述疫苗，和/或所述药物组合物。
另一方面，本申请提供了一种产生针对新型冠状病毒抗体的方法，其包括施用所述RNA、所述DNA、所述组合物、所述脂质体纳米颗粒制剂、所述疫苗，和/或所述药物组合物。
另一方面，本申请提供了一种激活免疫的方法，所述方法包括施用所述RNA、所述DNA、所述组合物、所述脂质体纳米颗粒制剂、所述疫苗，和/或所述药物组合物。
在某些实施方式中，所述方法为体外或离体的方法。
与现有技术相比，本发明的有益效果为：
1，本发明提供的三种mRNA都能在细胞内翻译，产生高水平的新型冠状病毒S蛋白，每种mRNA经由脂质体纳米颗粒包封后形成的制剂注射到小鼠体内，诱导小鼠产生高滴度中和抗体。与其他mRNA或未经密码子优化的mRNA相比，本申请的mRNA能够刺激更强的免疫反应，产生更高水平的中和抗体。
2，本发明提供的三种mRNA为CVG031、CVG032、CVG033。三种mRNA均包含编码新冠病毒S蛋白全长的开放阅读框，三种mRNA的开放阅读框均经过密码子优化，CVG031全长序列如SEQ ID NO.1，CVG032全长序列如SEQ ID NO.2所示，CVG033全长序列如SEQ ID NO.3所示；CVG031和CVG033编码相同的蛋白质序列，采用不同的密码子优化策略，编码的蛋s白质序列如SEQ ID NO.7所示；CVG032编码的蛋白质序列如SEQ ID NO.8所示。相比于Wuhan-Hu-1毒株S蛋白，Omicron毒株S蛋白的多个位点存在突变。本发明mRNA表达的蛋白质序列SEQ ID NO.7和SEQ ID NO.8均包含出现在新型冠状病毒Omicron毒株S蛋白中的多种突变，如A67V,Δ69-70,T95I,G142D,Δ143-145,Δ211,L212I,ins214EPE,G339D,S371L,S373P,S375F,K417N,N440K,G446S,S477N,T478K,E484A,Q493R,G496S,Q498R,N501Y,Y505H,T547K,D614G,H655Y,N679K,P681H,N764K,D796Y,N856K,Q954H,N969K,L981F；蛋白质序列SEQ ID NO.8还包含R682G、R683S、R685S突变。此外，以上SEQ ID NO.7蛋白序列含有S-2P突变，该突变 可使S蛋白构象稳定在融合前状态，稳定的融合前S蛋白S-2P突变比野生型S蛋白具有更好的免疫原性。所述的三种mRNA均含有5'Cap结构、5'非翻译序列(5'UTR)、3'非翻译序列(3'UTR)和poly(A)尾。三种mRNA序列中的结构单元U碱基全部被m1Ψ(1-methyl-3'-pseudouridylyl)替代。
附图说明
图1为本发明实施例2中mRNA在HEK 293T/17细胞中的表达结果。
图2为本发明实施例2中Western Blot检测mRNA转染HEK 293T/17细胞后的翻译产物。
图3为本发明实施例3中包被CVG031 mRNA的mRNA-LNP在室温(RT)和经过-80℃冻融后的粒径大小及均一性。
图4为本发明实施例3中包被CVG031 mRNA的mRNA-LNP在室温(RT)和经过-80℃冻融后的包封率。
图5为本发明实施例3中包被CVG031 mRNA的mRNA-LNP在室温(RT)和经过-80℃冻融后的表面电位属性。
图6为本发明实施例4中初次免疫一周后，小鼠血清中针对Omicron假病毒的中和抗体滴度；横坐标从左到右对应的依次是Saline、CVG031 3μg/只、CVG031 10μg/只、CVG031 30μg/只。
图7为本发明实施例4中经过两次免疫的一周后，小鼠血清中针对野生毒株假病毒、Delta假病毒、Omicron假病毒的中和抗体滴度，其中WT是指SARS-CoV-2野生毒株；横坐标从左到右对应的依次是Saline、CVG031 3μg/只、CVG031 10μg/只、CVG031 30μg/只。
图8为本发明实施例4中经过两次免疫的两周后，小鼠血清中针对野生毒株假病毒、Delta假病毒、Omicron假病毒的中和抗体滴度，其中WT是指SARS-CoV-2野生毒株；横坐标从左到右对应的依次是Saline、CVG031 3μg/只、CVG031 10μg/只、CVG031 30μg/只。
图9为CVG031诱导的中和抗体能与Omicron亚型发生交叉反应。
图10为CVG031引起CD4+T细胞(图10A-图10D)和CD8+T(图10E-图10H)细胞的抗原特异性反应，其中，野生型(图10A和图10E)、Delta(图10B和图10F)或Omicron (图10C和图10G)的Spike蛋白处理后IFNγ+/CD69+T细胞数量增加，delta S蛋白刺激的小鼠脾细胞中IL2+淋巴细胞显著增加(图10I)，同时，IL-5没有升高(图10J)。
图11为CVG031异源加强小鼠中对SARS-Cov-2 Delta和Omicron变体攻毒的保护作用，其中，A：Delta和Omicron变体挑战保护试验的实验设计，B和C：病毒感染后各组的存活率；D和E：感染后动物的体重变化。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“递送载体”通常是指能够将试剂(例如，mRNA)递送至靶细胞的转移媒介物。递送载体可以将试剂(例如，mRNA)递送到特定的细胞亚类。例如，借助递送载体的固有特征或者通过与载体相偶联的部分、包含在其内的部分(或者与载体结合的部分，从而使得该部分和该递送载体维持在一起，进而使得该部分足以靶向递送载体)使递送载体靶向某些类型的细胞。递送载体还可提高要递送的试剂(例如，mRNA)的体内半衰期和/或要递送的试剂的生物利用度。递送载体可包括病毒载体、病毒样颗粒、聚阳离子载体、肽载体、脂质体和/或杂交载体。例如，如果靶细胞是肝细胞，所述递送载体的性质(例如，尺寸、电荷和/或pH)可以有效地将所述递送载体和/或其中包载的分子(例如，mRNA)递送至靶细胞、降低免疫清除和/或促进在该靶细胞中停留。
在本申请中，术语“DNA”通常包括cDNA或基因组DNA，“RNA”通常包括mRNA，还包括使用核苷酸类似物(例如肽核酸和非天然存在的核苷酸类似物)产生的DNA或RNA的类似物，及其杂合体。DNA或RNA可以是单链或双链的。在本申请中，术语“mRNA”通常是指经过处理去除了内含子，且能够被翻译成多肽的RNA转录本。
在本申请中，术语“修饰”用于核酸(例如RNA或DNA)时通常是指，与相应的野生型相比，所述核酸具有不同的核苷酸分子，不同的核苷酸序列，由不同的键组成和/或在其结构中掺入非天然部分。例如，所述修饰可包括核苷酸的修饰，例如，所述核苷酸可包含经修饰的碱基、糖或磷酸基团。例如，所述修饰可包括不同的核苷酸序列但是编码相同氨基酸序列的多肽或蛋白质，或相同功能的多肽或蛋白质。所述修饰可以是化学修饰和/或生物修饰。“化学修饰”可包括引入不同于野生型或天然存在的核酸中所见到的那些化学物质的修饰，例如，共价修饰，例如，引入经修饰的核苷酸(例如，核苷酸类似物，或 者引入在这些核酸分子中未天然发现的侧基)。术语“经修饰的核苷酸”通常是指核酸聚合物中的单元，其含有经修饰的碱基、糖或磷酸基团，或在其结构中掺入非天然部分。
根据本发明，天然存在的或修饰核苷和/或核苷酸，或者优化的核苷酸可以用于制备修饰核酸，例如修饰mRNA。例如，修饰mRNA可包括一种或以上天然核苷(例如，腺苷、鸟苷、胞苷、尿苷)；修饰核苷(例如，2-氨基腺苷、2-硫代胸苷、肌苷、吡咯并嘧啶、3-甲基腺苷、5-甲基胞苷、C-5丙炔基-胞苷、C-5丙炔基-尿苷、2-氨基腺苷、C5-溴尿苷、C5-氟尿苷、C5-碘尿苷、C5-丙炔基-尿苷、C5-丙炔基-胞苷、C5-甲基胞苷、2-氨基腺苷、7-脱氮腺苷、7-脱氮鸟苷、8-氧代腺苷、8-氧代鸟苷、O(6)-甲基鸟嘌呤、假尿苷、(例如，N-1-甲基-假尿苷)、2-硫代尿苷以及2-硫代胞苷)；化学修饰碱基；生物修饰碱基(例如，甲基化碱基)；插入碱基；修饰糖(例如，2'-氟代核糖、核糖、2'-脱氧核糖、阿拉伯糖以及己糖)；修饰磷酸基基团(例如硫代磷酸基和5'-N-亚磷酰胺键)，或其任意组合。这些经过修饰的核苷酸可以是天然的核苷酸，也可以是经过人工优化或者改良的核苷酸。
RNA分子(例如，mRNA)可以包括至少0.1％的修饰核苷酸。修饰核苷酸的分数可以计算为：修饰核苷酸的数量/核苷酸的总数×100％。在一些实施例中，RNA分子包括约0.1％修饰核苷酸至约100％修饰核苷酸。在一些实施例中，RNA分子包括约0.1％修饰核苷酸、约0.2％修饰核苷酸、约0.5％修饰核苷酸、约1％修饰核苷酸、约2％修饰核苷酸、约5％修饰核苷酸、约10％修饰核苷酸、约20％修饰核苷酸、约50％修饰核苷酸、或约100％修饰核苷酸。
在本申请中，术语“密码子优化”用于核酸时，通常表示通过用在细胞中具有不同的相对使用频率的编码相同氨基酸残基的密码子，替换亲代多肽编码核酸中的一个、至少一个、或一个以上密码子，已经改良而在细胞中具有改善的表达的编码多肽的核酸，例如哺乳动物细胞或细菌细胞。
在本申请中，术语“药物组合物”通常是指以允许活性成分(例如，本申请的疫苗、组合物、药物组合物、载体、细胞、DNA或RNA)的生物学活性有效的形式的制剂，并且其不含有对所述制剂待施用的受试者有不可接受的毒性的另外成分。这些制剂可为无菌的。
在本申请中，术语“S蛋白”，也可称为“刺突蛋白”或“Spike蛋白”，通常是指冠状病毒表面的膜蛋白，其可在病毒表面形成突出的同型三聚体。
在本申请中，术语“疫苗”通常是指含有有效诱导受试者的抗特定病原体或疾病的治 疗程度的免疫性的活性组分的试剂或组合物。
在本申请中，术语“脂质纳米颗粒”通常是指包含通过分子间力彼此物理结合(例如，共价或非共价)的多个(即多于一个)脂质分子的颗粒。脂质纳米颗粒可以是例如微球(包括单层和多层囊泡，例如脂质体)、乳液中的分散相、胶团或悬浮液中的内相。脂质纳米颗粒可以包含一种或多种脂质(例如，阳离子脂质、非阳离子脂质和PEG-修饰的脂质)。
在本申请中，术语“脂质体”通常是指通过一个或多个双层的膜与外部介质隔离的具有内部空间的囊泡。例如，所述双层的膜可以通过两性分子形成，如包含空间隔离的亲水性和疏水性结构域的合成或天然来源的脂质；又例如，所述双层的膜可以通过两亲性聚合物和表面活性剂形成。
在本申请中，术语“新型冠状病毒”通常是指严重急性呼吸道综合征冠状病毒2型，英文全称为Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)。SARS-CoV-2属于冠状病毒科(Coronaviridae)乙型冠状病毒属(Betacoronavirus)沙贝病毒亚属(Sarbecovirus)。SARS-CoV-2是一种具有包膜的、不分节段的正链单股RNA病毒。其可引发新型冠状病毒肺炎(COVID-19)。
在本申请中，术语“药学上可接受的载体”通常是指任何与所施用的活性成分相容的溶剂、分散介质、涂层、等渗剂和吸收延迟剂等佐剂、赋形剂或其它药物载体。
在本申请中，术语“载体”旨在包括药学上可接受用于向相关动物施用或者如果适用的话可接受用于治疗或诊断目的的任何溶剂、分散介质、包衣、稀释剂、缓冲剂、等渗剂、溶液、悬浮液、胶体、惰性体等，或其组合。
在本申请中，术语“有效量”是指能够治疗或改善疾病或病况或者能够产生预期治疗效果的量。
用于本文时，术语“同源性”或“同一性”是指两个或更多个多核苷酸或多肽序列之间的互补性程度。当第一核酸或氨基酸序列具有与第二核酸或氨基酸序列完全相同的一级序列时，词语“同一性”可以代替词语“同源性”。序列同源性和序列同一性可通过使用本领域已知的算法和计算机程序分析两个或更多个序列来确定。这样的方法可用于评估给定序列是否与另一选定序列具有同一性或同源性。
在两个或更多个核酸或多肽序列的环境下，术语“同一”或“同一性”百分比是指当使用下面描述的序列比较算法之一(或普通技术人员可用的其他算法)或通过视觉检查测量进行比较和比对最大对应性时，两个或更多个序列或子序列相同或具有特定百分比的相 同的氨基酸残基或核苷酸。
在本文中，在说到“SEQ ID NO”表示的是DNA序列或RNA序列时，也包括与该序列互补的另外一条DNA序列或DNA序列。
在本文中，“SEQ ID NO.1-15”的含义具体如下表所示：
用于本文时，术语“试剂盒”可用于描述便携式自备外壳的变体，其包括至少一组本发明的试剂、组分或药学上配制的组合物。任选地，这样的试剂盒可包括一套或多套说明书，所述说明书关于，例如在实验室或临床应用中，使用所封装的组合物。
用于本文时，术语“预防”或“治疗”是指在疾病状态的临床症状发作之前施用单独的或包含在药物组合物中的化合物，以阻止所述疾病状态的任何症状、方面或特征。这样的预防和抑制不需要是绝对被认为是医学上有用的。
本申请还提供了如下的一种或多种实施方案：
1.编码新型冠状病毒S蛋白的RNA，其中所述新型冠状病毒S蛋白包含如SEQ ID NO.7-8中任一项所示的氨基酸序列。
2.如实施方案1所述的RNA，其为mRNA。
3.如实施方案1-2中任一项所述的RNA，其核苷酸序列是密码子优化的。
4.如实施方案1-3中任一项所述的RNA，其包含如SEQ ID NO.13-15中任一项所示的核苷酸序列。
5.如实施方案1-4中任一项所述的RNA，其包含选自下组的一种或多种结构：5'Cap结构、5'非翻译序列、开放阅读框、3'非翻译序列和poly(A)尾。
6.如实施方案5所述的RNA，其中所述poly(A)尾包含超过30个腺苷酸。
7.如实施方案5或6所述的RNA，其中，自5’端至3’端，所述poly(A)尾的结构为：30个腺苷酸、连接序列和70个腺苷酸。
8.如实施方案5-7中任一项所述的RNA，其中所述poly(A)尾包含SEQ ID NO.12所示的核苷酸序列。
9.如实施方案1-8中任一项所述的RNA，其包含如SEQ ID NO.1-3中任一项所示的核苷酸序列。
10.如实施方案1-9中任一项所述的RNA，其包含至少一种经修饰的核苷酸。
11.如实施方案1-10中任一项所述的RNA，其在选自下组的一个或多个位置处包含修饰：5'Cap结构、5'非翻译序列、开放阅读框、3'非翻译序列和poly(A)尾。
12.如实施方案10或11所述的RNA，其中所述经修饰的核苷酸包含一种或多种包含选自下组的核苷酸：N1-甲基假尿苷三磷酸(N1-Methylpseudo-UTP)、假尿苷三磷酸(pseudo-UTP)、5-甲氧基尿苷三磷酸(5-Methoxy-UDP)和5-甲基胞苷三磷酸(5-Methyl-CTP)。
13.转录实施方案1-12中任一项所述的RNA的DNA，所述DNA包含SEQ ID NO.9-11中任一项所示的核苷酸序列。
14.如实施方案13所述的DNA，其中所述DNA还包括5'非翻译序列、3'非翻译序列和poly(A)尾。
15.如实施方案13-14中任一项所述的DNA，其包含SEQ ID NO.4-6中任一项所示的核苷酸序列。
16.组合物，其包含实施方案1-12中任一项所述的RNA，和递送载体。
17.如实施方案16所述的组合物，其中所述递送载体包括脂质体。
18.如实施方案16-17中任一项所述的组合物，其中所述递送载体包括脂质纳米颗粒(LNP)。
19.如实施方案16-18中任一项所述的组合物，其中所述脂质纳米颗粒包括阳离子脂质、非阳离子脂质和胆固醇。
20.一种脂质体纳米颗粒制剂，其包被实施方案1-12中任一项所述的RNA。
21.一种疫苗，其包括实施方案1-12中任一项所述的RNA、实施方案13-15中任一项所述的DNA、实施方案16-19中任一项所述的组合物，和/或实施方案20所述的脂质纳 米颗粒制剂。
22.一种药物组合物，其包括实施方案1-12中任一项所述的RNA、实施方案13-15中任一项所述的DNA、实施方案16-19中任一项所述的组合物、实施方案20所述的脂质体纳米颗粒制剂、实施方案21所述的疫苗，和/或其药学上可接受的载体。
23.一种治疗和/或预防新型冠状病毒感染相关的疾病或病症的方法，所述方法包括向有需要的患者施用实施方案1-12中任一项所述的RNA、实施方案13-15中任一项所述的DNA、实施方案16-19中任一项所述的组合物、实施方案20所述的脂质体纳米颗粒制剂、实施方案21所述的疫苗，和/或实施方案22所述的药物组合物。
24.实施方案1-12中任一项所述的RNA、实施方案13-15中任一项所述的DNA、实施方案16-19中任一项所述的组合物、实施方案20所述的脂质体纳米颗粒制剂、实施方案21所述的疫苗，和/或实施方案22所述的药物组合物在制备药物中的用途，所述药物用于治疗和/或预防新型冠状病毒感染相关的疾病或病症。
25.载体，其包括实施方案1-12中任一项所述的RNA、实施方案13-15中任一项所述的DNA。
26.如实施方案25所述的载体，其包含病毒载体。
27.细胞，其包含实施方案1-12中任一项所述的RNA、实施方案13-15中任一项所述的DNA，和/或实施方案25-26中任一项所述的载体。
28.试剂盒，其包括实施方案1-12中任一项所述的RNA、实施方案13-15中任一项所述的DNA、实施方案16-19中任一项所述的组合物、实施方案20所述的脂质体纳米颗粒制剂、实施方案21所述的疫苗，和/或实施方案22所述的药物组合物。
29.产生针对新型冠状病毒抗体的方法，其包括施用实施方案1-12中任一项所述的RNA、实施方案13-15中任一项所述的DNA、实施方案16-19中任一项所述的组合物、实施方案20所述的脂质体纳米颗粒制剂、实施方案21所述的疫苗，和/或实施方案22所述的药物组合物。
30.激活免疫的方法，其包括施用实施方案1-12中任一项所述的RNA、实施方案13-15中任一项所述的DNA、实施方案16-19中任一项所述的组合物、实施方案20所述的脂质体纳米颗粒制剂、实施方案21所述的疫苗，和/或实施方案22所述的药物组合物。
31.如实施方案29-30中任一项中任一项所述的方法，其为体外或离体的方法。
实施例
下面结合实施例对本发明的技术方案进行详细说明。以下采用的试剂和生物材料如未特别说明，均为商业化产品。
本发明的具体实验过程为：1、获取SARS-CoV-2病毒的基因序列；2、获取SARS-CoV-2病毒的蛋白序列；3、设计编码抗原蛋白的DNA模板序列；4、体外转录产生mRNA；5、脂质体纳米颗粒包封mRNA；6、以包封mRNA的脂质体纳米颗粒制剂对小鼠进行免疫；7、检测小鼠血清的中和抗体。
实施例1 mRNA的制备
本发明所述的RNA进行序列优化后，使用本领域内技术人员公知的实验方法，将RNA体外转录的DNA模板克隆到pUC57载体，转化至大肠杆菌感受态细胞内，保存阳性克隆菌种。扩大培养菌种，抽取并纯化DNA模板质粒，质粒经酶切后线性化，以线性化质粒为模板进行RNA体外转录(IVT)，纯化RNA产物。具体步骤如下：
委托公司合成三种编码新冠病毒S蛋白全长的基因序列，基因序列经过密码子优化，序列如SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11所示，SEQ ID NO.9和SEQ ID NO.10翻译的蛋白质序列相同，如SEQ ID NO.7所示，SEQ ID NO.11翻译的蛋白质序列如SEQ ID NO.8所示。较佳地，三种基因与5'UTR DNA序列、3'UTR DNA序列、poly(A)尾共同合成。将包含5'UTR、开放阅读框、3'UTR、poly(A)尾的合成基因产物(序列分别如SEQ ID NO.4-6所示)克隆至pUC57载体。扩增载体后，以限制性核酸内切酶进行酶切，获得线性化载体。将纯化后的线性化载体作为RNA体外转录的模板，加入T7 RNA聚合酶、CTP、GTP、ATP、m1Ψ(1-methyl-3'-pseudouridylyl)修饰的UTP(即N1-甲基假尿苷三磷酸(N1-Methylpseudo-UTP))、Cap类似物((3'-OMe-m7G)(5')ppp(5')(2'-OMeA)pG，即m7G的3'羟基进行了甲基化修饰，确保了具有较高的翻译效率同时也具有抗反向转录的效果)，以及其他本领域技术人员所公知的必要成分，在37℃下孵育1-5小时。反应完毕以DNase进行消化，去除DNA，并进一步纯化获得mRNA产物。以本领域技术人员公知的方法检测mRNA浓度和完整性。制备得到三种mRNA，命名为CVG031、CVG032、CVG033，核酸序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示。
实施例2 mRNA细胞可译性检测
按照实施例1所述步骤制备mRNA，参考转染试剂产品指导，将CVG031 mRNA与转染试剂按指导比例混合，转染至HEK 293T/17细胞，检测mRNA转染细胞后的表达，如图1所示，图1显示了所述mRNA在HEK 293T/17细胞中的表达。通过Western Blot检测新冠病毒S蛋白全长表达，检测的S蛋白结果如图2所示。图2中的Spike指示新冠病毒S蛋白分子量大小，Ctrl是阴性对照组，没有观察到新型冠状病毒S蛋白的表达；包含CVG031 mRNA的LNP/CVG031组表达出新冠病毒S蛋白分子量相同大小的蛋白质产物。图1和图2结果表明：CVG031 mRNA能够翻译为新冠病毒的S蛋白。
实施例3脂质体纳米颗粒包被CVG031 mRNA
将实施例1获得的mRNA进行封装：将阳离子脂质(MC3)、DSPC、胆固醇和PEG-脂质以50:10:38.5:1.5的摩尔比溶解在乙醇中。以约10:1至30:1的总脂质与mRNA的重量比制备脂质纳米颗粒(LNP)。简而言之，实施例1的mRNA和四组分脂质混合物使用微流体纳米沉淀工艺制成mRNA-LNP，在该工艺中，酸性pH下的mRNA水溶液与脂质的乙醇溶液快速混合。然后通过切向流超滤(TFF)去除粗产物中的乙醇，然后使用PBS溶液(1x，pH 7.4)进行缓冲液交换。接下来，使用蔗糖溶液将中和产物中的mRNA稀释至0.5mg/mL。最后，通过0.22μm Sartopore PES膜对产品进行无菌过滤，随后将等分试样在室温下储存或在-80℃下冷冻(1.0mL填充)。
mRNA-LNP的表征：通过动态光散射(Malvern Nano ZS Zetasizer)测量mRNA-LNP的粒度、多分散性(PDI)和Zeta电位。直径用Z average表征。LNP中mRNA的封装效率(E.E.)定义为最终mRNA-LNP产物中封装的mRNA/总mRNA的质量比。使用特定的嵌入荧光染料(Quant-iTTM RNA Reagent)定量检测mRNA等核酸。根据使用mRNA标准生成的校准曲线计算mRNA浓度。mRNA-LNP中封装的mRNA的相对比例通过不存在与存在分散LNP的表面活性剂时的荧光信号的比率来确定。不存在表面活性剂时的信号表明游离mRNA的水平，而存在表面活性剂时的信号作为样品中总mRNA的量度。
包被CVG031 mRNA的mRNA-LNP在室温(RT)和经过-80℃冻融后的粒径大小及 均一性如图3所示，两种情况下的LNP直径几乎不变，均在60-80nm之间，说明CVG031 mRNA-LNP十分稳定。包被CVG031 mRNA的mRNA-LNP在室温(RT)和经过-80℃冻融后，对mRNA的包封率几乎都达到100％，如图4所示，说明本发明使用LNP制备的mRNA新冠疫苗制剂能够非常高效地将mRNA包裹在LNP内，并且经历-80℃冻融后仍然十分稳定。包被CVG031 mRNA的mRNA-LNP在室温(RT)和经过-80℃冻融后，纳米颗粒表面电位属性如图5所示，均保持负电荷属性，说明本发明的mRNA-LNP能够均匀地悬浮分布在溶液里；电荷绝对值较小，说明mRNA-LNP仍能较好通过外表面同样带有负电荷的细胞膜。
实施例4动物免疫
将实施例3包被CVG031 mRNA的脂质体纳米颗粒制剂过滤后，注射6-8周雌性Balb/c小鼠，对照组注射生理盐水(Saline)，脂质体纳米颗粒制剂的注射剂量分别3μg/只、10μg/只、30μg/只。在经过第一次免疫的七天后，采集小鼠血液，检测血清里的对Omicron新冠假病毒的中和抗体效价，结果如图6所示。初次免疫后的第21天，使用实施例3过滤后的脂质体纳米颗粒制剂再次注射小鼠，此为二次免疫。二次免疫一周后，采集小鼠血液，检测血清里的中和抗体效价，结果如图7所示。二次免疫两周后，采集小鼠血液，检测血清里的中和抗体效价，结果如图8所示。
实验结果表明：本发明提供的CVG031 mRNA与脂质体成分制成的脂质体纳米颗粒，注射至小鼠体内，诱导产生针Omicron突变型新冠病毒毒株的高水平的中和抗体，且中和抗体的水平随脂质体纳米颗粒浓度增加而升高。
核酸序列如SEQ ID NO.2和SEQ ID NO.3所示的CVG032mRNA和CVG033 mRNA，与CVG031mRNA具有相似的免疫效果，三种mRNA都能在细胞内翻译，产生高水平的新型冠状病毒S蛋白，每种mRNA经由脂质体纳米颗粒包封后形成的制剂注射到小鼠体内，诱导小鼠产生高滴度中和抗体。与其他mRNA或未经密码子优化的mRNA相比，本申请的三种mRNA能够刺激更强的免疫反应，产生更高水平的中和抗体。
实施例5 CVG031诱导的中和抗体与Omicron亚种的交叉反应
小鼠用用CVG031在第0天和第14天免疫两次，第二次免疫14天的动物血清样本与Omicron BA.1和BA.2的HIV假病毒孵育，并测试其中和活性。结果(图 9)显示，CVG031诱导的抗体与与Omicron BA.1和BA.2产生交叉反应。
实施例6 CVG031诱导细胞免疫
本实施例检测了CVG031免疫动物的T细胞免疫反应。用不同变体全长S蛋白再刺激所诱导的细胞因子进行细胞内染色，评价S蛋白特异性细胞免疫功能。接种方法同上，第二次接种后4周收集脾脏细胞进行分析。结果(图10)显示，CVG031可引起CD8和CD4T细胞的抗原特异性反应，这可以从分别用野生型、Delta或Omicron的Spike蛋白处理后IFNγ+/CD69+T细胞数量增加中看出(图10A-图10H)。结果表明，通过CVG031免疫小鼠，对新冠病毒产生了强烈的免疫反应。ELSA法检测S蛋白刺激的小鼠脾细胞中IL2的表达也显示出CVG031接种小鼠的IL2+淋巴细胞显著增加(图10I)。同时，IL-5在所有样本中均没有升高(图10J)，说明CVG031主要诱导的是特异性Th1免疫反应，而不是Th2。
实施例7 CVG031加强免疫
本实施例测试了BBIBP-CorV(中国生物集团北京生物制品研究所)加强同源BBIBP-CorV或异源CVG031加强针接种对经Delta或Omicron突变株感染的表达ACE2的转基因小鼠的保护作用。图11A显示，在完成2剂BBIBP-CorV接种后14天，动物注射了一次不同剂量的BBIBP-CorV或CVG031作为加强针。加强针注射后14天，这些动物通过鼻内吸收接种了200CCID50剂量的Delta或Omicron病毒量。在生理盐水对照组中，4/4的动物在感染Delta变异株后6天内死亡。与此同时，2/4的动物在感染Omicron病毒后8天内死亡，其余2只动物出现严重疾病(黑线，图11B和图11C)。在两种变异株感染后8天内，接种同源或异源免疫组均未出现动物死亡。然而，在BBIBP-CorV同源加强针实验组中，4/4只动物在感染Delta变异后第8天出现严重症状并出现显著体重减轻(灰色线，图11D)。
上述仅为本发明的部分优选实施例，本发明并不仅限于实施例的内容。对于本领域中的技术人员来说，在本发明技术方案的构思范围内可以有各种变化和更改，所作的任何变化和更改，均在本发明保护范围之内。

Claims (15)

  1. 编码新型冠状病毒S蛋白的RNA，其中所述新型冠状病毒S蛋白包含如SEQ ID NO.7或SEQ ID NO.8所示的氨基酸序列。
  2. 如权利要求1所述的RNA，其包含如SEQ ID NO.13-15中任一项所示的核苷酸序列。
  3. 如权利要求1-2中任一项所述的RNA，其包含下组的结构：5'Cap结构、5'非翻译序列、开放阅读框、3'非翻译序列和poly(A)尾。
  4. 如权利要求3所述的RNA，其中所述poly(A)尾包含SEQ ID NO.12所示的核苷酸序列。
  5. 如权利要求1-4中任一项所述的RNA，其包含如SEQ ID NO.1-3任一项所示的核苷酸序列。
  6. 如权利要求1-5中任一项所述的RNA，其包含至少一种经修饰的核苷酸。
  7. 如权利要求6所述的RNA，其中所述经修饰的核苷酸包含一种或多种包含选自下组的核苷酸：N1-甲基假尿苷三磷酸(N1-Methylpseudo-UTP)、假尿苷三磷酸(pseudo-UTP)、5-甲氧基尿苷三磷酸(5-Methoxy-UDP)和5-甲基胞苷三磷酸(5-Methyl-CTP)。
  8. 转录权利要求1-7中任一项所述的RNA的DNA，所述DNA包含SEQ ID NO.9-11所示的核苷酸序列。
  9. 如权利要求8所述的DNA，其包含下组的结构：5'非翻译序列、开放阅读框、3'非翻译序列和poly(A)尾。
  10. 如权利要求8-9中任一项所述的DNA，其包含SEQ ID NO.4-6中任一项所示的核苷酸序列。
  11. 一种脂质体纳米颗粒制剂，其包被权利要求1-7中任一项所述的RNA。
  12. 一种疫苗，其包括权利要求1-7中任一项所述的RNA、权利要求8-10中任一项所述的DNA，和/或权利要求11所述的脂质纳米颗粒制剂。
  13. 一种药物组合物，其包括权利要求1-7中任一项所述的RNA、权利要求8-10中任一项所述的DNA，和/或权利要求11所述的脂质纳米颗粒制剂、权利要求12所述的疫苗，和/或其药学上可接受的载体。
  14. 权利要求1-7中任一项所述的RNA、权利要求8-10中任一项所述的DNA，和/或权利要求11所述的脂质纳米颗粒制剂、权利要求12所述的疫苗，和/或权利要求13所述的药物组合物在制备药物中的用途，所述药物用于治疗和/或预防新型冠状病毒感染相关的疾病或病症。
  15. 根据权利要求14所述的用途，所述药物适于序贯施用。
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