WO2022068846A1 - Nouveau vaccin à arnm contre un coronavirus, sa méthode de préparation et son utilisation - Google Patents

Nouveau vaccin à arnm contre un coronavirus, sa méthode de préparation et son utilisation Download PDF

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WO2022068846A1
WO2022068846A1 PCT/CN2021/121536 CN2021121536W WO2022068846A1 WO 2022068846 A1 WO2022068846 A1 WO 2022068846A1 CN 2021121536 W CN2021121536 W CN 2021121536W WO 2022068846 A1 WO2022068846 A1 WO 2022068846A1
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seq
protein
mrna
sequence
present
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PCT/CN2021/121536
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Chinese (zh)
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胡勇
刘陈立
李楠
张昊
艾亮霞
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深圳市瑞吉生物科技有限公司
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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
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • 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
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the invention belongs to the technical field of genetic engineering, and specifically relates to a novel coronavirus (SARS-CoV-2) mRNA vaccine antigen design, a vaccine and a preparation method and application thereof.
  • SARS-CoV-2 novel coronavirus
  • Coronavirus is a kind of RNA virus that exists widely in nature. Coronaviruses are a large family with many members that infect only vertebrates and can cause respiratory, gastrointestinal and nervous system diseases in humans and animals. Novel coronavirus (SARS-CoV-2) is a new type of coronavirus discovered in 2019.
  • SARS-CoV-2 Before the discovery of SARS-CoV-2, there were 6 coronaviruses known to infect humans, 4 of which were relatively common in the population, were less pathogenic, and generally only caused mild respiratory symptoms similar to the common cold; the other two Viruses, SARS coronavirus and MERS coronavirus, are highly pathogenic. SARS-CoV-2 is highly contagious and can cause lung inflammation and, in severe cases, respiratory failure and death.
  • the structural proteins of the novel coronavirus consist of spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins.
  • S spike
  • E envelope
  • M membrane
  • N nucleocapsid
  • the S protein is presented on the surface of the virus in the form of a trimer, forming a "crown" spike structure, and mediating the binding of the virus to the cell receptor and membrane fusion.
  • the S protein is highly immunogenic, can activate T-cell immune responses, and can induce the production of neutralizing antibodies.
  • the S protein is an important target for current coronavirus vaccine and drug development. Several studies have shown that vaccines developed against SARS-CoV and MERS-CoV S protein can provide good immune protection.
  • the S protein Compared with other new coronavirus structural proteins, the S protein has a larger molecular weight and is more difficult to excrete from cells. By combining with a high-efficiency exocrine signal peptide, the exocytosis efficiency of the S protein can be effectively improved. At the same time, after sequence optimization, the expression of S protein in cells can be increased, and the expression of effective antigens can be enhanced.
  • the S protein coding sequence whose translation product is the prefusion conformation was obtained by inserting a Foldon trimerization domain at the C-terminus of the S protein.
  • the mRNA sequence corresponding to the S protein in the pre-fusion conformation can induce high titers of neutralizing antibodies in vivo with higher safety.
  • mRNA vaccine technology can be used as a fast and flexible technology platform to effectively deal with the threats of various emerging viruses. Therefore, mRNA vaccines are considered to be the most potential vaccines against the new coronavirus. Traditional inactivated vaccines, attenuated vaccines and peptide vaccines have long development cycles and complex production processes, making it difficult to meet the needs of public health emergencies. In addition, compared with DNA vaccines, mRNA vaccines have obvious advantages. First, mRNA vaccines are more effective than DNA vaccines. DNA vaccines need to pass through the cell membrane and nuclear membrane to express antigens, so when there is not enough dose of DNA to reach the nucleus, the vaccine will not be able to activate the immune system in the body and induce the production of antibodies.
  • mRNA vaccines are more biologically safe than DNA vaccines.
  • Foreign DNA has a certain chance of being integrated into the human genome, so DNA vaccines have the risk of causing genome mutations.
  • mRNA has no risk of gene integration into the genome.
  • mRNA is easily degraded after being translated into protein, and its transient expression feature not only ensures the safety of mRNA drugs, but also makes the dose controllable, avoiding antigen immune tolerance (no immune tolerance to specific antigens) caused by long-term exposure of vaccine drugs. the state of the reaction).
  • viruses of animal origin are not required to participate in the vaccine development process, the risks of using viruses and animals are avoided, and effective antigens can be obtained in a short period of time.
  • mRNA vaccines In general, compared with traditional vaccines, mRNA vaccines have a shorter production cycle, simpler production processes and lower costs. mRNA vaccines take only 6 weeks from antigen sequence design to production, while the production of traditional vaccines takes 5-6 months at the earliest. mRNA vaccines can be lyophilized and stored at room temperature for 6-18 months, while traditional vaccines need to be stored at 2-8°C. Therefore, mRNA vaccines can be stored longer, do not require a cold chain, and have lower transportation costs. Traditional vaccines are powerless against many new viruses, while mRNA vaccines are more applicable, and their sequences can be flexibly designed to deal with different pathogens.
  • One object of the present invention is to provide antigens for mRNA vaccines against novel coronavirus (SARS-CoV-2).
  • Another object of the present invention is to provide a vaccine against the novel coronavirus (SARS-CoV-2).
  • Another object of the present invention is to provide a method for preparing an mRNA vaccine against novel coronavirus (SARS-CoV-2).
  • Another object of the present invention is to provide mRNA for preparing a vaccine against novel coronavirus (SARS-CoV-2).
  • Another object of the present invention is to provide DNA for preparing the mRNA against the novel coronavirus (SARS-CoV-2).
  • Another object of the present invention is to provide a vector for preparing the mRNA against the novel coronavirus (SARS-CoV-2).
  • Another object of the present invention is to provide the application of mRNA vaccine against novel coronavirus (SARS-CoV-2).
  • the novel coronavirus presents a spherical ellipsoid with a diameter of 80-120 nm.
  • the virion surface has ball-and-stick protrusions composed of trimeric spike glycoproteins (Spike, S).
  • S trimeric spike glycoproteins
  • M membrane glycoproteins
  • E envelope proteins
  • N nucleocapsid
  • 2019-nCoV can utilize a tightly glycosylated, homotrimeric class I fusion bulge protein (S protein) to enter host cells.
  • S protein can exist in a relatively stable prefusion conformation and undergo Vigorous structural rearrangement promotes fusion of the viral membrane structure with the host cell membrane, a process induced by the binding of the viral S1 subunit to the host receptor, which can destabilize the prefusion trimer, resulting in the S1 subunit.
  • the unit falls off and prompts the S2 subunit to transition to a highly stable postfusion conformation.
  • the receptor-binding domain of S1 undergoes a chain-like conformational movement that temporarily hides or exposes the determinants of receptor binding, and these two states are considered "upward” " or "down” conformation, where the down conformation corresponds to the unreachable state of the receptor, and the up conformation corresponds to the receptor accessible state, the latter being considered unstable.
  • the amino acid sequence of the wild-type S protein was modified, and the viral antigen fragment encoded by the mRNA was designed, and then the encoded virus was synthesized.
  • mRNA of antigenic fragments to achieve immunity against novel coronavirus.
  • the present invention adopts genetic engineering technology to stabilize S in its prefusion conformation, preventing structural rearrangement, and exposing antigenic epitopes with better antigenicity can lead to higher neutralizing antibody responses.
  • the present invention respectively modifies the S protein coding sequence to stabilize the translation product in the pre-fusion conformation.
  • the relevant antigen sequences were cloned into plasmids by genetic engineering techniques for mRNA synthesis.
  • the present invention provides the following proteins of (a) or (b):
  • the protein composed of the amino acid sequence shown in SEQ ID No. 1 is the modified and improved protein on the basis of the wild-type S protein of the present invention, which is also referred to as the pre-fusion configuration S protein in the present invention.
  • the present invention designs and optimizes the S sequence, so that the expressed S protein is in the pre-fusion configuration, so as to achieve high-efficiency expression in human cells and increase immunogenicity.
  • the pre-fusion configuration of the S protein of the present invention is more likely to induce the production of neutralizing antibodies.
  • the amino acid sequence shown in SEQ ID No.1 is substituted, deleted and/or added with one or several amino acids and is composed of the amino acid sequence shown in SEQ ID No.1.
  • the protein has the same function as a derivative protein, at least comprising a sequence of at least 15 consecutive amino acids at the C-terminal of the amino acid sequence shown in SEQ ID No. 1.
  • the protein composed of the amino acid sequence shown in SEQ ID No. 1 having the same function means that the protein composed of the amino acid sequence shown in SEQ ID No. 1 has "more likely to induce the production of neutralizing antibodies” " and/or "increased immunogenicity” (compared to wild-type S protein).
  • the present invention also provides the use of the improved S protein (pre-fusion configuration S protein and/or its derivative protein) as an antigen in preparing a vaccine.
  • the present invention also provides DNA encoding the improved S protein of the present invention.
  • the nucleotide sequence of the DNA includes the sequence shown in SEQ ID No.10, SEQ ID No.11, SEQ ID No.12 or SEQ ID No.13.
  • the present invention also provides an expression vector, which contains the DNA of the present invention (DNA encoding the improved S protein of the present invention).
  • the present invention also provides mRNA encoding the improved S protein of the present invention.
  • the mRNA is obtained by in vitro transcription of the aforementioned DNA of the present invention.
  • the nucleotide sequence of the mRNA of the present invention includes the sequence shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5.
  • the mRNA of the present invention may further comprise an adjuvant sequence at the 5' end of its nucleotide sequence.
  • the adjuvant sequence can be a nucleotide sequence that promotes the secretory expression of a protein.
  • the adjuvant sequence comprises the sequence shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9.
  • the mRNA comprises the sequence shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 and SEQ ID No.2, SEQ ID No.9 respectively No.3, SEQ ID No.4 or SEQ ID No.5 combined sequence of mRNA (SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 any of the sequences shown at the 5' end of the sequence shown in any of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No. 5).
  • the mRNA comprises the sequence shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 combined with the sequence shown in SEQ ID No.4. mRNA shown.
  • the present invention also provides a method for preparing the mRNA.
  • the preparation method of the mRNA of the present invention comprises:
  • Synthesis of mRNA encoding viral antigen fragment Synthesize a DNA fragment encoding the pre-fusion conformational S protein (which can be synthesized by yourself or commercially), and clone it into a plasmid as a template for in vitro transcription (IVT) to obtain uncapped mRNA;
  • mRNA capping under the catalysis of capping enzyme, a cap structure is added to the 5' end of uncapped mRNA to obtain a capped mRNA molecule;
  • the capped mRNA is subjected to lithium chloride/ethanol precipitation, spin column, chlorine extraction/ethanol precipitation or gel purification to obtain purified mRNA.
  • the present invention also provides the application of the mRNA antigen design in the preparation of novel coronavirus mRNA vaccine.
  • the present invention also provides a vaccine comprising one or more of the mRNAs of the present invention.
  • the vaccine of the present invention is a preventive vaccine against a novel coronavirus (SARS-CoV-2), which may further include a PBS solution for dissolving mRNA.
  • SARS-CoV-2 novel coronavirus
  • the technology of the present invention is to first design the S protein sequence of the pre-fusion conformation in the new coronavirus (SARS-CoV-2) through bioinformatics methods to achieve high-efficiency expression in human cells and increase immunogenicity; and then synthesize through in vitro transcription methods mRNA encoding viral antigenic fragments to achieve immunity against novel coronavirus.
  • SARS-CoV-2 new coronavirus
  • the entire mRNA vaccine production cycle is short, the process is simple, the production cost is low, the storage time is long, no cold chain is required, and it is easy to transport.
  • Traditional vaccines cannot respond quickly to public health events caused by many new viruses, while mRNA vaccines are more applicable, and their sequences can be flexibly designed to deal with different pathogens. Rapid development of vaccines for acute infectious diseases plays an important role.
  • Figure 1 is a schematic diagram of the mRNA vaccine of the present invention.
  • Figure 2 shows the expression results of different mRNA vaccines encoding the S protein antigenic fragments in the pre-fusion configuration after sequence optimization in Example 1 of the present invention.
  • M protein marker
  • 1# S protein encoded by SEQ ID No.2 in the pre-fusion configuration
  • 2# S protein encoded by SEQ ID No.3 in the pre-fusion configuration
  • 3# encoded by SEQ ID No.4
  • the prefusion configuration S (prefusion) protein of 4# the prefusion configuration S (prefusion) protein encoded by SEQ ID No.5.
  • Figure 3A shows the results of exocytosis of 293T cells after 24 hours of transfection of mRNA (SEQ ID No. 4) with different adjuvant sequences (SEQ ID No. 6-9) in Example 2 of the present invention.
  • Control control group mRNA (SEQ ID No.4), 1#: mRNA with SEQ ID No.6 adjuvant sequence (SEQ ID No.4), 2#: with SEQ ID No.7 adjuvant sequence mRNA (SEQ ID No. 4), 3#: mRNA with SEQ ID No. 8 adjuvant sequence (SEQ ID No. 4), 4#: mRNA with SEQ ID No. 9 adjuvant sequence ( SEQ ID No. 4).
  • * represents p ⁇ 0.05 compared with the control group
  • *** represents p ⁇ 0.01 compared with the control group.
  • Figure 3B shows that mRNAs (SEQ ID No. 2-5) with different adjuvant sequences (SEQ ID No. 6-9) in another batch of experiments in Example 2 of the present invention were transfected into 293T cells 24 hours later. Exocrine results. * represents p ⁇ 0.05 compared with the control group, *** represents p ⁇ 0.01 compared with the control group.
  • Figure 4 shows the results of S-specific antibody titers after inoculating mice with different adjuvants in Example 3 of the present invention and the preferred solution mRNA (SEQ ID No. 4) in Example 2.
  • * represents p ⁇ 0.05 compared with the control group, *** represents p ⁇ 0.01 compared with the control group.
  • Figure 5A and Figure 5B show the results of neutralizing antibody titers after inoculating mice with adjuvanted mRNA (SEQ ID No. 4) in Example 4 of the present invention.
  • Figure 5A low dose group
  • Figure 5B high dose group.
  • Each curve in the figure represents one mouse.
  • Figure 5C shows the results of neutralizing antibody titers after inoculating mice with different doses of mRNA with different adjuvants in another batch of experiments in Example 4 of the present invention. * represents p ⁇ 0.01 compared to the control group.
  • Figure 6 shows that the mRNA with the mutated or deleted amino acid in Example 5 of the present invention also has the ability to produce neutralizing antibodies after inoculating mice. * represents p ⁇ 0.01 compared to the control group.
  • instruments, reagents, materials, etc. involved in the following examples are all conventional instruments, reagents, materials, etc. in the prior art, and can be obtained through regular commercial channels.
  • the experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are conventional experimental methods, detection methods, etc. existing in the prior art.
  • the DNA fragments used were commissioned synthesis.
  • the mRNA used was prepared as follows:
  • the specific sequence of the DNA fragment preferably includes The sequences shown in SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, and SEQ ID No.13, the 5' end of the DNA fragment can also include a corresponding adjuvant sequence (preferably SEQ ID No. 13) as needed. .6, the DNA fragment of SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9);
  • Capping of mRNA under the catalysis of a capping enzyme, a cap structure is added to the 5' end of an uncapped mRNA to obtain a capped mRNA molecule;
  • the capping enzyme includes but is not limited to dioxymethylation One or a combination of 2'-OMethyltransferase or RNA triphosphatase, RNA glutamyltransferase (guanyltransferase) and Guanine-7-methyltransferase (Guanine-7-methyltransferase) ;
  • the capped mRNA is subjected to lithium chloride/ethanol precipitation, spin column, chlorine extraction/ethanol precipitation or gel purification to obtain purified mRNA.
  • SEQ ID No. 1 S protein sequence in prefusion configuration
  • SEQ ID No.2 mRNA encoding the S protein sequence in the prefusion configuration (optimization scheme 1)
  • SEQ ID No.3 mRNA encoding the S protein sequence in the prefusion configuration (optimization scheme 2)
  • SEQ ID No.4 mRNA encoding the S protein sequence in the prefusion configuration (optimization scheme 3)
  • SEQ ID No.5 mRNA encoding the S protein sequence in the prefusion configuration (optimization scheme 4)
  • SEQ ID No. 10 DNA encoding the S protein sequence in the prefusion configuration
  • SEQ ID No. 11 DNA encoding the S protein sequence in the prefusion configuration
  • SEQ ID No. 12 DNA encoding the S protein sequence in the prefusion configuration
  • SEQ ID No. 13 DNA encoding the S protein sequence in the prefusion configuration
  • mRNAs SEQ ID No. 2-5) with different adjuvant sequences (SEQ ID No. 6-9) were transfected into 293T cells 24 hours later to extract the cell supernatant, see S in the supernatant protein exocytosis level. Specific steps are as follows:
  • wash the plate the coated 96-well plate, pour out the coating liquid, put it on the absorbent paper, and press the plate firmly until there is no residue in the well.
  • Blocking After washing the plate, dry the solution inside, add Blocking buffer in a volume of 250ul per well, then seal the plate with a sealing film, and seal at room temperature for 2 hours.
  • Serum or standard incubation Dilute the serum or standard (Yiqiao Shenzhou, 40591-V08H) in a gradient manner, add 100ul per well to the washed 96-well plate, then seal the plate and incubate at room temperature for 1.5 h.
  • Plate washing Complete the plate washing according to step 3, and the number of plate washing is increased to 6 times.
  • wash the board follows step 8 to complete the board washing. In this step, be sure to wash the board and dry the solution.
  • Termination Add 100ul of Stop buffer, read on the ELISA plate within 10 minutes, and set the absorption wavelength to 450nm.
  • Figure 3A shows the experimental results of mRNA (SEQ ID No. 4) with different adjuvant sequences (SEQ ID No. 6-9) in a batch of experiments.
  • Control control group mRNA (SEQ ID No.4), 1#: mRNA with SEQ ID No.6 adjuvant sequence (SEQ ID No.4), 2#: with SEQ ID No.7 adjuvant sequence mRNA (SEQ ID No. 4), 3#: mRNA with SEQ ID No. 8 adjuvant sequence (SEQ ID No. 4), 4#: mRNA with SEQ ID No. 9 adjuvant sequence ( SEQ ID No. 4).
  • the exocytosis effect of the pre-fusion conformation S protein encoded by the mRNA (SEQ ID No.4) with SEQ ID No.7 adjuvant sequence is significantly higher than that with SEQ ID No.6/SEQ ID No.8/SEQ ID
  • the S protein in the prefusion configuration encoded by the mRNA of No. 9 adjuvant sequence (SEQ ID No. 4).
  • Figure 3B shows the exocytosis results of 293T cells transfected with mRNAs (SEQ ID No. 2-5) with different adjuvant sequences (SEQ ID No. 6-9) for 24 hours in another batch of experiments.
  • the four groups from left to right in the figure are the SEQ ID No.2 group with different adjuvant sequences (SEQ ID No.6-9) and the SEQ ID No.2 groups with different adjuvant sequences (SEQ ID No.6-9).
  • Set of ID No. 3 Set of SEQ ID No. 4 with different adjuvant sequences (SEQ ID No. 6-9), SEQ ID No. 5 with different adjuvant sequences (SEQ ID No. 6-9)
  • the leftmost histogram in each group represents the mRNA of the Control control group (that is, the leftmost histogram in the four groups respectively represents SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 ).
  • mRNAs SEQ ID No. 4
  • adjuvant sequences of SEQ ID No. 6-9 were injected into 6-week-old balb/c mice twice on the 1st day and the 14th day, respectively.
  • the mouse serum was collected on the 14th and 35th day, and the titer of anti-S protein-specific antibody in the serum was detected.
  • Coating Dilute the S protein (Yiqiao Shenzhou, 40591-V08H) with coating buffer to a solution of 200ng/ml, add it to the ELISA plate, the volume of each well is 100ul, and each dilution is repeated 3 times The wells were covered with sealing film and coated overnight at 4°C.
  • wash the plate the coated 96-well plate, pour out the coating liquid, put it on the absorbent paper, and press the plate firmly until there is no residue in the well.
  • Blocking After washing the plate, dry the solution inside, add Blocking buffer in a volume of 250ul per well, then seal the plate with a sealing film, and seal at room temperature for 2 hours.
  • Serum incubation Dilute mouse serum with dilution buffer to 40x, 400x, 4000x, 40000x, 400000x, 4000000x, 40000000x, add 100ul per well to the washed 96-well plate, then seal the plate at room temperature Incubate for 1.5h.
  • Plate washing Complete the plate washing according to step 3, and the number of plate washing is increased to 6 times.
  • step 8 Wash the board: Following step 8 to complete the board washing. In this step, be sure to wash the board and dry the solution.
  • Termination Add 100ul of Stop buffer, read on the ELISA plate within 10 minutes, and set the absorption wavelength to 450nm.
  • Control control group mRNA (SEQ ID No. 4), 1#: mRNA with SEQ ID No. 6 adjuvant sequence (SEQ ID No. 4), 2#: with SEQ ID No. 6 mRNA with SEQ ID No. 7 adjuvant sequence (SEQ ID No. 4), 3#: mRNA with SEQ ID No. 8 adjuvant sequence (SEQ ID No. 4), 4#: with SEQ ID No. 8 9 Adjuvant Sequence mRNA (SEQ ID No. 4).
  • the mRNA (SEQ ID No. 4) with the wild-type S protein mRNA (30ug/piece) and the adjuvant sequence with SEQ ID No. 7 and SEQ ID No. 1ug/mice) and high dose (30ug/mice) were inoculated into 6-week-old balb/c mice by two injections on the 1st day and the 14th day, and the mouse serum was collected on the 35th day, through focus reduction neutralization tests (FRNT) Neutralizing antibody titers were measured experimentally. Incubate at 37°C for 1 h by mixing standard doses of 2019-nCoV with two-fold serial dilutions of inactivated serum at 56°C.
  • the virus-serum mixture (100 ⁇ l) was then inoculated into cultured 293T ACE2 overexpressing cells. After 3 days of incubation, plates were formalin-fixed and permeabilized by sequential incubation with biotin-conjugated S protein monoclonal antibody, streptavidin-HRP, and TrueBlue peroxidase substrate (KPL). , closed and stained. The number of foci in the positive control group without serum was the highest. When the foci count in the experimental group was less than or equal to 50%, it was taken as the neutralization critical value, and the geometric mean was calculated as the technical replicate.
  • Figure 5A mRNA with SEQ ID No. 9 adjuvant sequence (SEQ ID No. 4) low-dose group (1ug/only)
  • Figure 5B with SEQ ID No. .9 Adjuvant sequence mRNA (SEQ ID No. 4) high-dose group (30ug/only).
  • Figure 5C shows the mRNA with SEQ ID No. 7 or SEQ ID No. 9 adjuvant sequence compared to the wild-type S protein (WT) high-dose treatment group (30ug/head) in another batch of experiments (SEQ ID No.4) low-dose group (1ug/only), mRNA with SEQ ID No.7 or SEQ ID No.9 adjuvant sequence (SEQ ID No.4) high-dose group (30ug/only) Specific neutralizing antibodies were produced.
  • the neutralizing antibody titer was proportional to the dose of the candidate vaccine, and the protective effect of neutralizing antibody in the high-dose (30ug/vaccine) group was significantly better than that in the low-dose (1ug/vaccine) group.
  • the mRNA (SEQ ID No.4 and mutant D614G, L56V, 144del, N501Y, 69del/70del) with the adjuvant sequence of SEQ ID No. 6-week-old balb/c mice were inoculated twice on the 14th day, and the mouse serum was collected on the 35th day, and the neutralizing antibody titer was detected by the focus reduction neutralization tests (FRNT).
  • -nCoV was mixed with two-fold serial dilutions of 56°C inactivated serum and incubated at 37°C for 1 hour. The virus-serum mixture (100 ⁇ l) was then inoculated into cultured 293T ACE2 overexpressing cells.

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Abstract

La présente invention concerne un nouveau vaccin à ARNm contre un coronavirus, sa méthode de préparation et son utilisation. La présente invention concerne une protéine parmi les suivantes (a) ou (b) : (a) une protéine constituée de la séquence d'acides aminés telle que représentée dans la SEQ ID NO : 1 ; et (b) une protéine dérivée qui est obtenue par substitution, délétion et/ou addition d'un ou plusieurs acides aminés dans la séquence d'acides aminés telle que représentée dans la SEQ ID NO : 1 et a la même fonction que la protéine constituée de la séquence d'acides aminés telle que représentée dans la SEQ ID NO : 1. La présente invention conçoit une séquence de protéine S d'une conformation de pré-fusion dans un nouveau coronavirus (SRAS-CoV-2), ce qui permet d'obtenir une expression à haut rendement de cellules humaines et d'augmenter l'immunogénicité. L'ARNm de la présente invention peut réaliser une immunité contre le nouveau coronavirus, et présente une large applicabilité.
PCT/CN2021/121536 2020-09-29 2021-09-29 Nouveau vaccin à arnm contre un coronavirus, sa méthode de préparation et son utilisation WO2022068846A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113736801A (zh) * 2020-05-28 2021-12-03 上海蓝鹊生物医药有限公司 mRNA及包含其的新冠病毒mRNA疫苗
CN117330750A (zh) * 2023-12-01 2024-01-02 北京生物制品研究所有限责任公司 一种筛选新冠病毒早期毒种的方法和制造疫苗的方法

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