WO2022144040A1 - Séquence nucléotidique codant pour un nouvel antigène de coronavirus, et son utilisation - Google Patents
Séquence nucléotidique codant pour un nouvel antigène de coronavirus, et son utilisation Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Definitions
- the present disclosure relates to the technical field of vaccines, in particular to a nucleotide sequence encoding a novel coronavirus antigen and applications thereof, in particular to the application in the preparation of vaccines.
- Coronaviruses are an enveloped non-segmented positive RNA virus belonging to the family Coronaviridae and the order Nidovirales, and are the largest known positive-strand RNA viruses. According to the serological and genomic characteristics of the virus, the subfamilies of coronaviruses can be divided into four genera, ⁇ , ⁇ , ⁇ , and ⁇ . The pneumonia of unknown cause that occurred in Wuhan in December 2019 was finally determined to be caused by a new type of coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2). It is spread through respiratory droplets and can also cause pneumonia (Novel Coronavirus-infected Pneumonia, NCP) through contact transmission, and the population is generally susceptible.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus
- CN111218459B discloses a new coronavirus vaccine with human replication-deficient adenovirus type 5 as a carrier, which can induce the body to produce cellular and humoral immune responses in a short time, and has good immune protection effect.
- the new crown vaccine in this patent is a viral vector vaccine.
- Nucleic acid vaccines are called “third-generation vaccines" and have the following advantages: 1. All-round induction of humoral and cellular immune responses, which can play a good preventive role; 2. Simple production process, good storage stability, and no need for cold chain Transport, suitable for large-scale application and distribution. At present, relevant nucleic acid vaccines exist.
- patent CN110951756B discloses a nucleic acid sequence expressing SARS-CoV-2 virus antigen peptide, which can be effectively expressed in human cells and induce corresponding immune protection responses, which can be developed into SARS-CoV-2 2
- the vaccine which ensures the uniqueness of protein expression by removing potential alternative splicing sites, reduces the difficulty of subsequent purification of the protein, and further optimizes its codons by reducing the GC content to obtain the optimized nucleic acid sequence. Vaccines with high expression levels.
- the present disclosure provides a nucleotide sequence for encoding the S protein of a novel coronavirus (SARS-CoV-2), and the nucleotide sequence can be used to prepare a corresponding nucleic acid vaccine.
- SARS-CoV-2 novel coronavirus
- the nucleic acid vaccine described in the present disclosure can increase the expression of its antigenic protein in vivo, and stimulate a more efficient immune response, thereby improving the preventive and/or therapeutic effect on SARS-CoV-2 virus.
- the present disclosure provides a nucleotide sequence encoding a novel coronavirus antigen.
- the nucleotide sequence includes the coding sequence for the SARS-CoV-2 virus S protein (SARS-CoV-2 virus surface protein Spike).
- the coding sequence of the S protein of the SARS-CoV-2 virus has homology with the nucleotide sequence of the wild-type S protein, and the nucleotide sequence of the wild-type S protein is as shown in SEQ ID NO:1 shown.
- the coding sequence of the S protein of the SARS-CoV-2 virus has 65-80% homology with the nucleotide sequence of the wild-type S protein.
- the coding sequence of the S protein of the SARS-CoV-2 virus has 70-75% homology with the nucleotide sequence of the wild-type S protein.
- the third base of the codon in the coding sequence of the S protein of the SARS-CoV-2 virus is replaced by A with C or G, and T with C or G.
- the GC content of the coding sequence of the S protein of the SARS-CoV-2 virus is 40-80%.
- the GC content of the coding sequence of the S protein of the SARS-CoV-2 virus is 45-70%, optionally 50-60%.
- the coding sequence of the S protein of the SARS-CoV-2 virus is shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
- the nucleotide sequence further includes a signal peptide sequence as shown in SEQ ID NO: 2.
- the expression of the post-translation protein can be further increased by enhancing the transport of the post-translation protein between organelles, thereby enhancing the immunogenicity of the nucleic acid vaccine.
- the signal peptide sequence of the unoptimized S protein wild-type is shown in SEQ ID NO:10.
- the nucleotide sequence is shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.
- amino acid sequence of the S protein of the SARS-CoV-2 virus is shown in SEQ ID NO:9.
- the present disclosure also provides a vector comprising the above-mentioned nucleotide sequence.
- the vector includes, but is not limited to, plasmid, virus, phage, RNA, and optionally plasmid DNA.
- the vector is capable of expressing the SARS-CoV-2 virus S protein, which is capable of eliciting an immune response in mammals.
- the present disclosure also provides the application of the above-mentioned nucleotide sequence or vector in the preparation of SARS-CoV-2 nucleic acid vaccine.
- the present disclosure also provides a SARS-CoV-2 nucleic acid vaccine comprising the above-mentioned nucleotide sequence or vector.
- the present disclosure also provides applications of the above-mentioned nucleotide sequences, vectors or SARS-CoV-2 nucleic acid vaccines in the preparation of medicines for preventing and/or treating SARS-CoV-2 virus-related diseases.
- the present disclosure improves the GC content in the nucleotide sequence by optimizing the nucleotide sequence encoding the SARS-CoV-2 surface protein Spike, and further increases the GC content at the 5' end of the nucleotide; Codon frequency, increase the CAI index (codon adaptation index); increase the proportion of degenerate codon tails G or C, reduce the free energy of forming RNA secondary structure, reduce the proportion of Negative CIS elements, and reduce the proportion of repetitive sequences in the sequence; and By optimizing the signal peptide of the wild-type gene, the expression amount can be further increased, the optimized nucleotide sequence can be obtained, and the expression amount of the post-translation protein amount can be increased, thereby enhancing the immunogenicity of the nucleic acid vaccine.
- nucleic acid vaccine which greatly improves the gene transcription and expression of antigenic proteins compared with the wild-type sequence, and can induce more efficient humoral and cellular immune responses after immunizing experimental animals.
- the optimized nucleotide sequence is inserted into a eukaryotic expression vector, and then introduced into a host cell, so that the viral Spike antigen can be highly expressed in the host cell and on the surface.
- Viral humoral and cellular immune responses Antibodies produced by an activated humoral immune response can prevent virus entry, an activated cellular immune response can further clear virus-infected cells, and an activated cellular immune response can reduce potential side effects caused by "antibody-dependent enhancement" (ADE) adverse reactions.
- ADE antibody-dependent enhancement
- Figure 1 is the result of enzyme digestion verification of the nucleic acid vaccine candidate engineering bacteria plasmid in Example 2 of the disclosure, wherein A is the BamHI/EcoRV digestion result of the wild-type DNA plasmid pVAX1-S (WT), and B is the DNA plasmid pVAX1- The BamHI/XhoI digestion result of ADV400, C is the BamHI/XhoI digestion result of the DNA plasmid pVAX1-ADV401, and D is the BamHI/XhoI digestion result of the DNA plasmid pVAX1-ADV402;
- A is the BamHI/EcoRV digestion result of the wild-type DNA plasmid pVAX1-S (WT)
- B is the DNA plasmid pVAX1-
- C is the BamHI/XhoI digestion result of the DNA plasmid pVAX1
- the bands are as follows: 1. pVAX1-S (WT), 2. pVAX1-S (WT) after digestion, 3. pVAX1-ADV400, 4. pVAX1-ADV400 after digestion, 5. pVAX1-ADV401, 6. The digested pVAX1-ADV401, 7. pVAX1-ADV402, and 8. the digested pVAX1-ADV402.
- Figure 2 is a graph showing the qPCR transcription detection of the disclosed candidate nucleic acid vaccine in mammalian cells, wherein A is the detection result of pVAX1-ADV400, B is the detection result of pVAX1-ADV401, and C is the detection result of pVAX1-ADV402.
- FIG. 3 is a diagram showing the expression detection of mammalian cell antigen protein of candidate nucleic acid vaccines of the present disclosure, wherein A is a flowchart of flow detection, B is a flow detection result chart, and C is a flow detection result statistical chart.
- Figure 4 is a diagram showing the detection of the humoral immune response of the disclosed candidate nucleic acid vaccine, wherein A is the detection result of pVAX1-ADV400, B is the detection result of pVAX1-ADV401, and C is the detection result of pVAX1-ADV402.
- Figure 5 is an ELISPOT detection chart of the disclosed candidate nucleic acid vaccine cellular immune response, wherein A is the detection result of pVAX1-ADV400, B is the detection result of pVAX1-ADV401, and C is the detection result of pVAX1-ADV402.
- A is a flow cytometry flow chart
- B is a flow cytometry result graph
- C is a flow cytometry result statistical graph.
- SARS-CoV-2 in this article refers to "new coronavirus”.
- S protein herein refers to "SARS-CoV-2 virus surface protein Spike (spike protein)”.
- SFU spot-forming unit
- nucleic acid sequence optimization is: (1) According to the preference of the host cell for nucleic acid codons, degenerate codons are optimized, so that the optimized sequence contains more nucleic acid codons that are conducive to host cell recognition; (2) In On the basis of codon preference optimization, the GC content in the nucleic acid sequence is further optimized, so that the sequence after GC content optimization can express more target proteins; (3) The nucleic acid sequence is optimized so that it can transcribe more stable mRNA, which is beneficial to the target protein. protein translation; (4) changing the codon frequency of the host preference and increasing the CAI index (codon adaptation index).
- Optimization purpose to increase the protein expression of the target protein in host cells.
- Optimization strategy Optimize the third base of amino acid-encoding codons from A to C or G, or T to C or G; increase the ratio of G or C at the tail of degenerate codons, and reduce the free energy of forming RNA secondary structure, Reduce the proportion of Negative CIS elements and reduce the proportion of repetitive sequences in the sequence.
- Optimization step Select the SARS-CoV-2 virus surface protein Spike (S protein) as the antigen, and its amino acid sequence is shown in SEQ ID NO: 9, and the wild-type nucleic acid coding sequence SWT (SEQ ID NO. : 1) carry out optimization to obtain the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, and combine the optimized sequence with the optimized signal peptide sequence (SEQ ID NO: 5). : 2) connect to obtain 3 nucleotide sequences named ADV400 (SEQ ID NO: 6), ADV401 (SEQ ID NO: 7) or ADV402 (SEQ ID NO: 8) respectively, and then carry out the optimized sequence synthesis.
- S protein S protein
- ADV400, ADV401, and ADV402 sequences increased the GC content at the 5' end of DNA (60%), while the GC content at the same site of the wild-type sequence was less than 50%.
- the 3 nucleotide sequences obtained above, and the wild-type coding sequence of the S protein were transformed and constructed into the pVAX1 carrier (ThermoFisher, article number: V26020), respectively, to obtain plasmid DNAs: pVAX1-S (WT), pVAX1- ADV400, pVAX1-ADV401 and pVAX1-ADV402.
- Example 2 The plasmid DNA solutions prepared in Example 1 (volume not exceeding 10 ⁇ L): pVAX1-S (WT), pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV402 were added to the competent cells, and shaken gently. , placed on ice for 30min.
- the candidate plasmids pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV402 extracted in step 2 were digested with BamHI/XhoI double enzymes for specific restriction fragment identification, and pVAX1-S (WT) was specifically digested with BamHI/EcoRV double enzymes
- the restriction enzyme digestion fragment was identified; the restriction enzyme digestion system is as shown in Table 1, and the digestion reaction was carried out at 37°C for 4 hours. After digestion, electrophoresis was performed on a 1% agarose gel.
- nucleic acid vaccines prepared by the optimized nucleotide sequences ADV400, ADV401 and ADV402 in the present disclosure namely pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV402, compared with wild
- the nucleic acid vaccine pVAX1-S (WT) prepared with the nucleotide sequence of the same type, and the empty plasmid pVAX1 have significantly increased transcriptional effects in mammalian cells.
- the staining buffer is 2% FBS/PBS, and the staining volume is 50 ⁇ L per well. Gently blow and suck 3-5 times. Dyeing at room temperature in the dark for 15 min.
- the detection results are shown in FIG. 3 .
- the nucleic acid vaccines prepared from the optimized nucleotide sequences ADV400, ADV401 and ADV402 in the present disclosure namely pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV402
- the expression of antigenic protein in mammalian cells was significantly increased.
- the prepared nucleic acid vaccine was used to immunize mice twice every two weeks. The first day of vaccine immunization was counted as day 0. At the same time, buffer solution was used as a control, which was recorded as SSC.
- S peptide S antigen-specific peptide pool MHC-I/MHC-II epitope peptide
- Enzyme-linked immunosorbent assay for humoral immune response blood samples were collected from mice on the 14th day, and the specific antibody titers of the serum were determined by ELISA. When it is necessary to quantitatively detect the antibody concentration in mouse serum, a standard curve is added on the basis of conventional ELISA, and the concentration of antibody in mouse serum is determined according to the concentration of the standard curve.
- the detection results are shown in Figure 4, and it can be seen from Figure 4 that the optimized nucleotide sequences ADV400, ADV401 and ADV402 in the present disclosure, the prepared nucleic acid vaccines, namely pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV402, Compared with the nucleic acid vaccine pVAX1-S(WT) prepared with wild-type nucleotide sequence, the concentration of antibody in the serum of mice was significantly increased.
- Detection of cellular immune responses by immune cell-specific stimulation performed in a sterile environment, the mice were de-necked, euthanized, the spleen or lymph nodes were removed, and ground into a single-cell suspension; cells were harvested by centrifugation, and the red blood cell lysate was resuspended and then lysed with FBS. PBS to terminate the lysis; filter, count the prepared single cell suspension, and plate with 1 ⁇ 10 6 cells/well; add the corresponding specific polypeptide pools according to ELISPOT and flow cytometry respectively for in vitro stimulation, ELISPOT at 37°C, Detection was performed after 24h incubation in 5% CO 2 . Flow assay was cultured at 37°C, 5% CO 2 for 6 h, and the stimulated cells were collected by centrifugation. Flow cytometry detection.
- the ELISPOT detection results are shown in Figure 5, and the flow detection results are shown in Figure 6, indicating that the optimized nucleotide sequences ADV400, ADV401 and ADV402 in the present disclosure, the prepared nucleic acid vaccines, namely pVAX1-ADV400, pVAX1- Compared with the nucleic acid vaccine pVAX1-S(WT) prepared with wild-type nucleotide sequence, the specificity of Spike antigen was significantly increased for ADV401 and pVAX1-ADV402.
- the present disclosure improves the GC content in the nucleotide sequence by optimizing the nucleotide sequence encoding the SARS-CoV-2 surface protein Spike, and further increases the GC content at the 5' end of the nucleotide; at the same time, changing the host Preferential codon frequency increases the CAI index; increases the proportion of degenerate codon tails G or C, reduces the free energy of forming RNA secondary structures, reduces the proportion of Negative CIS elements, and reduces the proportion of repetitive sequences in the sequence.
- the optimized gene signal peptide can be further improved, and the optimized nucleotide sequence can be obtained and made into a nucleic acid vaccine.
- nucleic acid vaccine prepared by the optimized nucleotide sequence greatly improves the gene transcription and expression of the antigenic protein compared with the wild-type sequence, and can induce more efficient humoral and cellular immune responses after immunizing experimental animals. .
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Abstract
L'invention concerne un nucléotide codant pour un nouvel antigène de coronavirus, et son utilisation. Le nucléotide est obtenu par optimisation d'une séequence d'ADN de type sauvage codant pour une protéine Spike de surface du virus du SARS-CoV-2, et un peptide signal de gène de type sauvage, après avoir été optimisé, est relié aunucléotide en amont et inséré dans un vecteur d'expression eucaryote, puis introduit dans une cellule hôte, pour exprimer efficacement un antigène Spike de virus dans la cellule hôte. Après extraction d'un antigène, une réponse immunitaire humorale antivirale et une réponse immunitaire cellulaire sont systématiquement activées.
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CN113185586B (zh) * | 2021-05-18 | 2023-01-17 | 深圳市因诺转化医学研究院 | SARS-CoV-2编码蛋白来源的T细胞表位多肽及其应用 |
CN113046370B (zh) * | 2021-05-31 | 2021-09-07 | 北京华芢生物技术有限公司 | 新型冠状病毒b.1.1.7英国突变株rbd的基因及其应用 |
CN113249408B (zh) * | 2021-06-23 | 2021-11-02 | 深圳湾实验室 | 一种靶向激活体液免疫和细胞免疫的核酸疫苗载体构建及应用 |
CN113521272B (zh) * | 2021-07-28 | 2023-05-09 | 四川大学 | 新型冠状病毒肺炎dna纳米疫苗及其制备方法 |
CN113480619B (zh) * | 2021-08-26 | 2023-05-09 | 深圳市亚辉龙生物科技股份有限公司 | 多肽及其在新型冠状病毒检测中的应用 |
CN113528549B (zh) * | 2021-09-17 | 2021-12-21 | 艾棣维欣(苏州)生物制药有限公司 | 编码新型冠状病毒b.1.351突变株抗原的dna分子、dna疫苗及应用 |
CN113862286B (zh) * | 2021-12-03 | 2022-03-04 | 艾棣维欣(苏州)生物制药有限公司 | 编码sars-cov-2病毒c.37突变株抗原的dna分子、dna疫苗及应用 |
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