Multi-antigen monkeypox RNA vaccine and preparation method thereof

Abstract

The present invention relates to a multi-antigen monkeypox RNA vaccine and a preparation method thereof, and to the technical field of nucleic acid vaccines. The multi-antigen mRNA comprises a sequence encoding an antigenic polypeptide of a monkeypox virus or an antigenic fragment, variant or derivative thereof, and the antigenic polypeptide or antigenic fragment thereof at least comprises viral proteins of an intracellular mature virus and an extracellular enveloped virus of the monkeypox virus. The present invention provides a multi-antigen mRNA vaccine and sequence for monkeypox virus, the mRNA of the present invention can express antigenic proteins in in vitro cells, and after immunizing mice, high-titer anti-monkeypox virus A29L, A33L, B6R, M1R and E8L protein IgG can be detected, and high-titer anti-cowpox Tiantan strain live virus neutralizing antibodies are produced, which has good immunogenicity and is of great significance for the prevention of monkeypox virus.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid vaccines, and in particular to multiple mRNAs and multi-antigen monkeypox virus mRNA vaccines containing the same and a preparation method thereof. Background Art

[0002] The global popularization of safe and effective vaccines is the most powerful weapon to stop the monkeypox virus and the key to ending the monkeypox epidemic. So far, there is no specific vaccine for monkeypox. Monkeypox virus, smallpox virus and cowpox virus belong to the same orthopox virus. Smallpox vaccine can provide cross-protection. Vaccination with smallpox vaccine is 85% effective in preventing monkeypox or reducing the severity of infection. Currently, there are only two vaccines approved by the FDA for the prevention of monkeypox virus: ACAM2000 and JYNNEOS. However, these vaccines can cause rare side effects, such as myocarditis and pericarditis, and have a higher impact on patients with eczema and pregnant women. Therefore, there is still a need to develop a new generation of effective and safe monkeypox-specific vaccines. And to this day, monkeypox virus is still rampant, and the public health crisis caused by monkeypox virus has not been resolved.

[0003] Monkeypox virus is an enveloped double-stranded DNA virus with two different genetic evolutionary branches - the Central African branch and the West African branch. The West African branch has a case fatality rate of about 3.6%, while the Central African branch has historically caused more severe disease, with a case fatality rate of about 10.6%, and is considered more contagious.

[0004] mRNA vaccines have technical advantages such as short R&D cycle and fast production capacity growth, and are more suitable for emergency research and development of preventive vaccines for monkeypox virus. The development of an independent and controllable multi-antigen mRNA vaccine that can prevent monkeypox infection is of great significance to the prevention and control of monkeypox epidemics. Summary of the invention

[0005] The object of the present invention is to enrich the types of monkeypox virus vaccines in the prior art, and to provide a multi-antigen mRNA vaccine, a pharmaceutical composition and a kit for monkeypox virus. The design difficulty of the mRNA vaccine of monkeypox virus is relatively high, especially the realization of virus integrity and efficient expression will be affected by various factors. The mRNA provided by the present invention can express antigenic proteins in cells in vitro, and high titer anti-vaccinia Tiantan strain live virus neutralizing antibodies can be detected after immunizing mice, which has good immunogenicity and is of great significance for the prevention of monkeypox virus.

[0006] The technical solution provided by the present invention is as follows:

[0007] In a first aspect, the present invention provides an mRNA comprising an antigenic polypeptide encoding a multi-antigen monkeypox virus or an antigenic fragment, variant or derivative thereof, wherein the antigenic polypeptide or antigenic fragment thereof comprises at least viral proteins of the intracellular mature virion (IMV) and the extracellular enveloped virion (EEV) of the monkeypox virus.

[0008] In the present invention, the infectious disease Monkeypox (MPX) is a viral zoonosis caused by infection with Monkeypox virus (MPXV), which can be transmitted from animals to humans across species and can be transmitted from person to person. IMVs and EEVs express different viral proteins, forming unique antigenic features to distinguish the two forms of virus particles.

[0009] In one embodiment, the antigenic polypeptide or antigenic fragment thereof comprises mature virus particle surface proteins A29L, M1R, E8L and enveloped virus particle surface proteins A35R and B6R with protective immunogenicity. In the present invention, the A29L protein is the monoclonal antibody target of MPXV, mediating the recognition of host cells so that the virus catalyzes the fusion with the host cells. A35R is an important component of enveloped virus particles and is also a potential detection target for serology. The B6R protein is present on the membrane of EEV particles, negatively regulates complement activation, and its interaction with A33 or A34 determines its intracellular localization. E8L has a ganglioside binding motif composed of subsites, and the ganglioside binding domain of the viral protein is a characteristic target of antiviral drugs and neutralizing antibodies. The combination of intracellular mature virus particles and extracellular enveloped virus particles can provide better protection.

[0010] In the present invention, the multi-antigen mRNA at least simultaneously contains mRNA sequences encoding A29L, M1R, E8L, A35R and B6R of monkeypox virus.

[0011] In one embodiment, the nucleotide sequence of the mRNA is one of the sequences shown in SEQ ID No.1 to SEQ ID No.6, or a sequence that has more than 90% homology with one of the sequences shown in SEQ ID No.1 to SEQ ID No.6 and has the same function.

[0012] In the present invention, the CDS of the monkeypox virus mRNA is composed of optimized codons, and can efficiently express proteins at the cellular level (high levels of proteins are expressed after transfection into cells), and the proteins in the sequences shown in SEQ ID No. 1 to SEQ ID No. 6 can all be efficiently expressed. After the mRNA of the present invention is prepared into a vaccine (a vaccine expressing monkeypox antigen protein), it is verified by mouse immunization that the vaccine has good effectiveness and immunogenicity.

[0013] In a preferred embodiment, the nucleotide sequence of the mRNA has more than 95% homology with one of the sequences shown in SEQ ID No. 1 to SEQ ID No. 6 and has the same function.

[0014] In a more preferred embodiment, the nucleotide sequence of the mRNA has more than 99% homology with one of the sequences shown in SEQ ID No. 1 to SEQ ID No. 6 and has the same function.

[0015] The "homology" described in the present invention is synonymous with "identity", and means that in terms of the use of amino acid sequences or nucleotide sequences, the used sequences have (including but not limited to) 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% similarity compared with the sequences obtained in the prior art, and still have the same function as the original amino acid sequence or nuclear sweet potato sequence.

[0016] In one embodiment, the nucleotide sequence of the mRNA encoding the A29L, M1R, E8L, A35R and B6R domains of the monkeypox virus protective immunogenic protein is as shown in SEQ ID No.1 to SEQ ID No.6.

[0017] The mRNA includes the following elements in order from 5' to 3' direction: 5' cap structure, 5' UTR sequence, antigenic polypeptide of monkeypox virus or antigenic fragment, variant or derivative thereof, 3' UTR sequence and poly A tail sequence.

[0018] The mRNA-2949 DNA sequence is as described in SEQ ID No. 1: from the 5' end, positions 1 to 52 are 5'-UTR, positions 49-52 are Kozak sequences, positions 53 to 121 are secretory peptides, positions 122 to 493 are CDS-A35R (encoding amino acids 1-124 of monkeypox virus A35R protein), positions 494 to 508 are flexible linkers, positions 509 to 1048 are CDS-M1R (encoding amino acids 1-180 of monkeypox virus M1R protein), positions 1049 to 1066 are flexible linkers, and positions 1067 to 1900 are CDS-B6R (encoding amino acids 1-180 of monkeypox virus M1R protein). The first two sequences are CDS-A29L (encoding amino acids 1-278 of monkeypox virus B6R), the first two sequences are CDS-A29L (encoding amino acids 1-109 of monkeypox virus A29L), the first two sequences are CDS-B8L (encoding amino acids 1-244 of monkeypox virus E8L), the first two sequences are stop codons; the first two sequences are 3'-UTR, and the first two sequences are Poly A tails.

[0019] The mRNA-3000 DNA sequence is shown in SEQ ID No. 2: from the 5' end, positions 1 to 52 are 5'-UTR, positions 49-52 are Kozak sequences, positions 53 to 121 are secretory peptides, positions 122 to 493 are CDS-A35R (encoding amino acids 1-124 of monkeypox virus A35R protein), positions 494 to 508 are flexible linkers, positions 509 to 1048 are CDS-M1R (encoding amino acids 1-180 of monkeypox virus M1R protein), positions 1049 to 1114 are 2A short peptides, and positions 1115 to 1951 are CDS-B6R (encoding monkeypox virus The first two sequences are CDS-A29L (encoding amino acids 1-279 of monkeypox virus B6R protein), the first two sequences are CDS-A29L (encoding amino acids 1-109 of monkeypox virus A29L protein), the first two sequences are CDS-E8L (encoding amino acids 1-244 of monkeypox virus E8L protein), the first two sequences are CDS-E8L (encoding amino acids 1-244 of monkeypox virus E8L protein), the first two sequences are stop codons; the first two sequences are 3053-3178 for 3'-UTR, and the first two sequences are 3179 to 3298 for Poly A tail.

[0020] mRNA-2949A DNA template sequence (SEQ ID No.3):

[0021] From the 5' end, positions 1 to 52 are 5'-UTR, positions 49-52 are Kozak sequences, positions 53 to 121 are secretory peptides, positions 122 to 661 are CDS-M1R (encoding amino acids 1-180 of monkeypox virus M1R protein), positions 662 to 676 are flexible linkers, positions 677 to 1048 are CDS-A35R (encoding amino acids 1-124 of monkeypox virus A35R protein), positions 1049 to 1066 are flexible linkers, and positions 1067 to 1900 are CDS-B6R (encoding monkeypox virus B The first two sequences are CDS-A29L (encoding amino acids 1-279 of monkeypox virus A29L protein), the first two sequences are CDS-E8L (encoding amino acids 1-244 of monkeypox virus E8L protein), the first two sequences are CDS-E8L (encoding amino acids 1-244 of monkeypox virus E8L protein), the first two sequences are stop codons; the first two sequences are 3002-3127 and the first two sequences are 3'-UTR and the first two sequences are Poly A tail.

[0022] mRNA-3000A DNA template sequence (SEQ ID No.4):

[0023] From the 5' end, the 1st to 52nd are 5'-UTR, the 49th to 52nd are Kozak sequences, the 53rd to 121st are secretory peptides, the 122nd to 661st are CDS-M1R (encoding amino acids 1-180 of monkeypox virus M1R protein), the 662nd to 676th are flexible linkers, the 667th to 1048th are CDS-A35R (encoding amino acids 1-124 of monkeypox virus A35R protein), the 1049th to 1114th are 2A short peptides, the 1115th to 1951st are CDS-B6R (encoding amino acids 1-124 of monkeypox virus B6 R protein), 1952 to 1969 are flexible linkers, 1970 to 2296 are CDS-A29L (encoding amino acids 1-109 of monkeypox virus A29L protein), 2297 to 2317 are flexible linkers, 2318 to 3049 are CDS-E8L (encoding amino acids 1-244 of monkeypox virus E8L protein), 3050 to 3052 are stop codons; 3053-3178 are 3'-UTR, and 3179 to 3298 are Poly A tails.

[0024] mRNA-2949B DNA template sequence (SEQ ID No.5):

[0025] From the 5' end, the 1st to 52nd are 5'-UTR, the 49th to 52nd are Kozak sequences, the 53rd to 121st are secretory peptides, the 122nd to 661st are CDS-M1R (encoding amino acids 1-180 of monkeypox virus M1R protein), the 662nd to 676th are flexible linkers, the 667th to 1003rd are CDS-A29L (encoding amino acids 1-109 of monkeypox virus A29L protein), the 1004th to 1021st are flexible linkers, the 1022nd to 1393rd are CDS-A35R (encoding amino acids 1-109 of monkeypox virus A35L protein), the 1393rd to 1504th are CDS-A40R (encoding amino acids 1-109 of monkeypox virus A40L protein), the 1393rd to 1504th are CDS-A55R (encoding amino acids 1-109 of monkeypox virus A40L protein), the 1393rd to 1504th are CDS-A40 ... The first two sequences are CDS-B6R (encoding amino acids 1-279 of monkeypox virus B6R protein), the first two sequences are CDS-E8L (encoding amino acids 1-244 of monkeypox virus E8L protein), the first two sequences are stop codon, the first two sequences are 3002-3127 and 3128-3247 are poly A tail.

[0026] mRNA-22949C DNA template sequence (SEQ ID No.6):

[0027] From the 5' end, the 1st to 52nd are 5'-UTR, the 49th to 52nd are Kozak sequences, the 53rd to 121st are secretory peptides, the 122nd to 661st are CDS-M1R (encoding amino acids 1-180 of monkeypox virus M1R protein), the 662nd to 676th are flexible linkers, the 667th to 1003rd are CDS-A29L (encoding amino acids 1-109 of monkeypox virus A29L protein), the 1004th to 1021st are flexible linkers, the 1022nd to 1393rd are CDS-A35R (encoding amino acids 1-109 of monkeypox virus A35L protein), the 1393rd to 1504th are CDS-A40R (encoding amino acids 1-109 of monkeypox virus A40L protein), the 1393rd to 1504th are CDS-A55R (encoding amino acids 1-109 of monkeypox virus A40L protein), the 1393rd to 1504th are CDS-A40 ... The first two sequences are CDS-E8L (encoding amino acids 1-244 of monkeypox virus E8L protein), the first two sequences are CDS-B6R (encoding amino acids 1-279 of monkeypox virus B6R protein), the first two sequences are stop codons; the first two sequences are 3002-3127 and 3128-3247 are poly A tails.

[0028] In addition, the kozak sequence can be replaced by GCC, ACC, GCCACC or GCCANN; the stop codon can be replaced by TAA, TAG, or a combination of two stop codons such as TGA, TAA, and TAG can be added; the PolyA tail can be more than 30 A in length, or the 31st to 40th A can be replaced by GCATATGACT. The linker can be replaced, such as a CDS encoding an amino acid sequence such as GSGSAS, (GGGGS)n (n=1-5), GSAGSA, and AAAGGAA.

[0029] In a third aspect, the present invention provides a biological material related to the aforementioned nucleic acid molecule or protein, wherein the biological material comprises any one of the following B1) to B6):

[0030] B1) a nucleic acid molecule encoding the protein, wherein the nucleic acid molecule is a DNA molecule encoding the aforementioned multiple mRNAs;

[0031] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0032] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0033] B4) a recombinant plasmid containing the nucleic acid molecule described in B1), or a recombinant plasmid containing the expression cassette described in B2);

[0034] B5) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0035] B6) A transgenic cell line containing the nucleic acid molecule described in B1), or a transgenic cell line containing the expression cassette described in B2), or a transgenic cell line containing the recombinant vector described in B3).

[0036] In the present invention, the coding sequence of the protein can be cloned into a plasmid by genetic engineering technology, and in vitro transcription can be performed for mRNA synthesis; preferably including: 1) cloning the DNA fragment corresponding to the mRNA into the expression plasmid to obtain a recombinant plasmid; 2) transferring the recombinant plasmid into a host cell to obtain a recombinant cell, extracting the plasmid from the expanded recombinant cell, and performing PCR amplification to obtain a DNA template for in vitro expression of mRNA; 3) constructing an RNA in vitro synthesis system including the DNA template to perform in vitro synthesis of mRNA to obtain the active ingredient mRNA. In the present invention, the specific sequence of the DNA fragment can be determined according to the principle of complementary base pairing.

[0037] In one embodiment, after in vitro transcription, the transcribed RNA product needs to be subjected to a capping reaction, and the 5' end of the obtained mRNA is connected with a cap (Cap-1) structure.

[0038] In one embodiment, the recombinant vector is vector pVAX1.

[0039] In one embodiment, the recombinant vector is transferred into cells expressing the viral protein for expression. Preferably, the cells are selected from HEK293T cells, 293FTX cells, and HEK293A cells.

[0040] In a fourth aspect, the present invention further provides an mRNA-lipid complex, wherein the mRNA-lipid complex comprises a delivery vector and the aforementioned mRNA;

[0041] Preferably, the delivery vehicle comprises any one of ionizable liposomes, cationic liposomes, ionizable proteins, cationic proteins, ionizable polymers, cationic polymers, ionizable micelles, cationic micelles, ionizable lipid nanoparticles, and cationic lipid nanoparticles;

[0042] More preferably, the delivery vehicle is selected from ionizable lipid nanoparticles (LNPs).

[0043] In one embodiment, the mRNA-lipid complex is selected from ionizable lipid-mRNA complexes, cationic lipid-mRNA complexes or novel cationizable lipid-mRNA complexes, ionizable lipid-mRNA lipid nanoparticles, cationic lipid-mRNA lipid nanoparticles or novel cationizable lipid-mRNA lipid nanoparticles;

[0044] Preferably, the ionizable lipid-mRNA complex, cationic lipid-mRNA complex or novel cationizable lipid-mRNA complex further comprises protamine, PEGylated lipid, 1,2-dioleyl-sn-glycero-3-phosphoethanolammonium and / or cholesterol.

[0045] Preferably, the ionizable lipid-mRNA complex nanoparticle, cationic lipid-mRNA lipid nanoparticle or novel cationizable lipid-mRNA lipid nanoparticle further comprises PEGylated lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol.

[0046] In one embodiment, the mRNA-lipid complex includes lipid nanoparticles LNP-mRNA prepared by combining mRNA with ionizable lipids, and cationizable lipid material-mRNA lipid nanoparticles prepared by combining mRNA with ionizable lipid SM102.

[0047] In one embodiment, the preparation method of the mRNA-lipid complex comprises: mixing the mRNA with an ionizable lipid material and then packaging it with lipids; wherein the ionizable lipid material can be YK009, MC3, SM102, ALC0315, Lipid5, DOTAP, etc.;

[0048] In a preferred embodiment, the preparation method comprises dissolving an ionizable lipid material, 1,2-distearoyl-sn-glycero-3-phosphocholine and DMG-PEG2000, and then mixing the mixture, and then mixing mRNA with the lipid material.

[0049] In a fifth aspect, the present invention provides a multi-antigen monkeypox virus mRNA vaccine, comprising the aforementioned mRNA, the aforementioned biological material, or the aforementioned mRNA-lipid complex;

[0050] Preferably, the mRNA vaccine induces cells to produce virus-like particles; and / or, the mRNA vaccine also includes an adjuvant.

[0051] The term "adjuvant" refers to an agent that increases, stimulates, activates, enhances or modulates the immune response to the active ingredients of the composition at the cellular level or humoral level.

[0052] The inventors have conducted a large number of experiments and finally found that the specific combination of the framework sequence and the coding sequence of the present invention can enable the prepared vaccine to achieve better immunogenicity and stability.

[0053] The multi-antigen monkeypox virus mRNA vaccine is a vaccine against monkeypox virus, and its administration subjects include but are not limited to mammals and humans. The mammals include but are not limited to monkeys, camels, cattle, horses, goats, sheep, pigs, cats, dogs, rabbits, mice or rats. Preferably, the vaccine is an infectious disease vaccine for preventing infection with monkeypox virus. The "prevention" described in the present invention refers to all behaviors that avoid symptoms or delay the tension of specific symptoms by administering the product described in the present invention before or after the disease begins to develop.

[0054] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the aforementioned mRNA, the aforementioned biomaterial or the aforementioned mRNA-lipid complex and / or the aforementioned multi-antigen mRNA vaccine, and optionally a pharmaceutically acceptable carrier. The present invention provides the use of a product comprising the aforementioned mRNA, the aforementioned biomaterial or the aforementioned mRNA-lipid complex and / or the aforementioned mRNA vaccine in the preparation of a medicament for preventing and / or treating monkeypox virus infection.

[0055] In a seventh aspect, the present invention provides a kit comprising the aforementioned mRNA, the aforementioned biological material or the aforementioned mRNA-lipid complex, the aforementioned mRNA vaccine and / or the aforementioned pharmaceutical composition.

[0056] In the present invention, the term "neutralizing antibody" generally refers to the fact that after microorganisms invade the human body, they will stimulate the production of many types of antibodies, but only some antibodies can quickly identify microorganisms and "catch" them before they invade human cells, protecting the human body from infection. This process is called neutralization, and the antibodies that play a role are neutralizing antibodies.

[0057] In the present invention, the vaccine active ingredients mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000A, mRNA-2949B and mRNA-2949C are prepared from ionizable lipids to form lipid nanoparticles LNP-mRNA vaccine preparations that can achieve in vitro cellular monkeypox virus immunogenic antigen protein expression; the vaccine active ingredients mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000A, mRNA-2949B and mRNA-2949C are prepared from cationizable lipid SM102 to form lipid nanoparticles LNP-mRNA vaccine preparations that can achieve in vitro cellular monkeypox virus immunogenic antigen protein expression.

[0058] The beneficial effects of the present invention are:

[0059] The successful development of mRNA vaccines depends to a large extent on the optimization of the mRNA sequence itself. The mRNA provided by the present invention as the active ingredient of the vaccine is composed of a 1083 backbone code sequence and different antigen coding sequences. The mRNA sequence is codon-optimized mRNA, and is transfected into HEK293T cells using a commercial transfection reagent package. It was found that the mRNA can simultaneously express a large amount of antigen proteins in the cells, that is, the cells have good antigen expression efficiency in vitro.

[0060] The multi-antigen mRNA vaccine provided by the present invention can achieve stable and safe expression in vivo and effectively activate immune response, and can induce neutralizing antibody response. High titer of neutralizing antibodies against live vaccinia Tiantan strain virus with an average NT of 1.0 50They were mRNA-2949: ~1:2323, mRNA-3000: ~1:2185, mRNA-2949A: ~1:5938, mRNA-3000A: ~1:5042, mRNA-2949B: ~1:5053 and mRNA-2949C: ~1:6163, respectively. The neutralizing antibody levels induced by mRNA-2949A and mRNA-2949C were higher than those in other groups, indicating that mRNA-2949A and mRNA-2949C had good immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Schematic diagram of the structures of the active ingredients mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000A, mRNA-2949B and mRNA-2949C of the monkeypox virus mRNA vaccine provided by the present invention;

[0062] Figure 2 is a diagram of the mass analysis result in Example 1 of the present invention;

[0063] Figure 3 This is a diagram showing the results of the cell transfection protein expression detection in Example 1 of the present invention;

[0064] Figure 4 Detection of specific antibody IgG in serum of mice after immunization in Example 2 of the present invention;

[0065] Figure 5 The neutralizing antibody NT of the immune mouse serum against the live vaccinia virus of Tiantan strain in Example 2 of the present invention 50 Detection result diagram of

[0066] Figure 6 The reaction system formula for the in vitro transcription step in Example 1;

[0067] Figure 7 This is the reaction system formula for the capping reaction step in Example 1. DETAILED DESCRIPTION

[0068] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0069] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0070] Example 1. Sequence design, preparation and in vitro cell antigen expression detection of monkeypox virus mRNA vaccine

[0071] 1. Sequence design of monkeypox mRNA vaccine

[0072] The sequence design of the monkeypox mRNA vaccine uses an optimized mRNA backbone code sequence to enhance the stability of mRNA and the efficiency of protein expression. The CDS of the monkeypox virus mRNA is composed of optimized codons, which determines the amino acid sequence of the monkeypox virus IMV and EEV proteins that protect immunogenicity.

[0073] To achieve in vitro transcription of mRNA, the monkeypox virus mRNA vaccine sequence template was constructed on the vector PVAX1 (the sequence between the last three bases GGG of the T7 promoter and the multiple cloning site XhoI was replaced with the mRNA template sequence, and the BsaI restriction site sequence was inserted between the mRNA template sequence and the XhoI restriction site to obtain a recombinant plasmid, while other sequences remained unchanged). The T7 promoter sequence was used to initiate in vitro transcription under the action of T7 transcriptase.

[0074] Thus, the monkeypox mRNA vaccine active components mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000A, mRNA-2949B and mRNA-2949C were designed and optimized. Figure 1 The sequences of mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000A, mRNA-2949B and mRNA-2949C are shown in the sequence table as 1, 2, 3, 4, 5 and 6, respectively.

[0075] 2. In vitro synthesis of monkeypox virus mRNA vaccine

[0076] 1. The designed monkeypox virus mRNA vaccine sequence was synthesized as a DNA template and cloned into the PVAX1 vector to obtain the mRNA template DNA plasmid.

[0077] 2. Transform the template DNA plasmid into competent cells DH5α, and obtain a large number of amplified bacteria by culturing the host E. coli. Use the endotoxin-free plasmid extraction kit (Tiangen Biotechnology (Beijing) Co., Ltd., DP117) to extract the recombinant plasmid amplified in the amplified bacteria.

[0078] The amplified recombinant plasmid was linearized: the extracted recombinant plasmid was linearized by enzyme digestion with Bsa I, and the template that could be used for in vitro mRNA synthesis was obtained after purification, and the quantification was performed using the Qubit™ dsDNA BR Assay Kit (Invitrogen, Q32850).

[0079] 3. Use the linearized DNA prepared in step 2 as the template for in vitro synthesis of mRNA and prepare Figure 6 The reaction system shown was incubated at 37°C for 3 h for in vitro transcription to obtain a large amount of in vitro transcribed RNA, which was quantified using the Qubit™ dsDNA BR Assay Kit (Invitrogen, Q32850).

[0080] 4. Purify the in vitro transcribed RNA product in step 3: Add 1 μL RNase-FreeDNase I to the transcription reaction system and incubate at 37°C for 15 minutes to remove the DNA template in the in vitro transcription product system to obtain the transcription product. Then purify the obtained transcription product as follows:

[0081] (1) Add RNase-Free H 2 O to make up the volume to 200 μL;

[0082] (2) Add 200 μL of mixed solution A (water-saturated phenol: chloroform: isoamyl alcohol, v:v:v, 25:24:1), vortex for 10 seconds, centrifuge at 13,800 × g for 5 minutes at 4°C, and then transfer the upper aqueous phase in the tube to a new tube;

[0083] (3) Add an equal volume of mixed solution B (chloroform:isoamyl alcohol, v:v, 24:1) to the new tube, vortex for 10 seconds, centrifuge at 13,800 × g for 5 minutes at 4°C, and then transfer the upper aqueous phase from the tube to the new tube;

[0084] (4) Add an equal volume of 5 M ammonium acetate solution to a new tube, vortex to mix, place on ice for 15 min, centrifuge at 4°C, 13,800 × g for 15 min, and discard the supernatant.

[0085] (5) After adding 70% ice-cold ethanol to clean up the RNA, discard the 70% ethanol; add an appropriate amount of RNase-Free water (Solarbio, R1600) to resuspend, and use the Qubit™ RNA BR Assay Kit (Invitrogen, Q10211) kit for quantification.

[0086] 5. Perform mRNA capping reaction on the RNA transcription and purification product obtained in step 4. The specific steps are as follows:

[0087] (1) RNA denaturation: Take 60 µg of purified transcription product, incubate at 65°C for 15 min for denaturation treatment, and then move to ice.

[0088] (2) mRNA capping reaction: After adding the RNA denaturation product, follow Figure 7 The reaction system was prepared as shown, and incubated at 37° C. for 0.5 h to obtain a capped mRNA product having a Cap 1 structure, wherein Cap 1 is methylguanosine.

[0089] 6. Purification of mRNA capping product: Same as step 4.

[0090] 7. mRNA quality analysis:

[0091] The quality of the synthesized mRNA was analyzed using the Qsep100 fully automated nucleic acid and protein analysis system and RNA Cartridge Kit (C105110). The specific steps are as follows:

[0092] (1) mRNA denaturation: Dissolve mRNA in 1× Dilution Buffer, denature at 70°C for 2 min, and then immediately place on ice.

[0093] (2) Prepare buffer and buffer: Place enzyme-free sterile water in positions P, W, and C according to the instructions, and place Separation Buffer in position S. Place RNA Low Marker in well MC1.

[0094] (3) Insert and calibrate the card: Open the card door on the top of the instrument, insert the card, and close the card door. Click Latch to lock the card, click Tool Recalibrate to recalibrate the card, select the appropriate Voltage and Alignment Marker type in the pop-up option box, click Start Calibration, and the Calibration Succeed window will pop up when the calibration is completed. Click OK.

[0095] (4) Program setting: Click the blank box below Sample Position to pop up the 96-well plate simulation diagram. Select the sample position on the left. Double-click the corresponding position to enter the sample information. Click OK after the setting is completed. Click the blank box below Method to pop up the test method box. Select the appropriate Alignment Marker and Method. Click OK after completion. After the program is set, click Run to start electrophoresis separation.

[0096] (5) Data calculation and analysis

[0097] The results are as follows Figure 2As shown, the results showed that the in vitro synthesized monkeypox mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000A, mRNA-2949B and mRNA-2949C ( Figure 2 The bands shown in the table were consistent with the target bands, and the concentrations were 1758ng / μL, 1824ng / μL, 2150ng / μL, 1879ng / μL, 2083ng / μL and 1994ng / μL, respectively.

[0098] 3. Detection of protein expression by cell transfection of mRNA

[0099] 1. Inoculation of cells: 293T cells (ATCC) were inoculated in 12-well plates, 3×10 cells per well. 5 cells, 37°C, 5% CO 2 Incubate the cells in an incubator until they reach 80-90% confluence and then transfection can be performed.

[0100] 2. Prepare transfection complex: using TransIT ® -mRNA Transfection Kit (MIR, 2250): 100μL Opti-MEM + 1μg mRNA, add 2μL TransIT-mRNA Reagent and 2μL TransIT-mRNA Boost after mixing, then mix and stand at room temperature for 4 minutes to form a transfection complex.

[0101] 3. Transfect cells: Add the transfection complex to the cells and shake up and down to make the transfection complex evenly distributed. Incubate at 37°C and 5% CO 2 The cells were harvested after incubation for 18 h. There was no need to replace the cell culture medium before and after transfection.

[0102] 4. Extract total cell protein: After washing the cells twice with PBS, use cell lysis buffer RIPA (Kinple, P06M11) + protease inhibitor 100× (Kinple, P01C01), vortex to fully lyse the cells. After ice bathing for 30 minutes, centrifuge at 4°C at 13800×g for 15 minutes and take the supernatant.

[0103] 5. Quantification of total cell protein: Use BCA protein quantification kit (Jinple, P06M16) to quantify the total protein in the cell lysis supernatant. Mix the cell lysis supernatant with BCA working solution, incubate at 37°C for 45 minutes, detect the absorbance at A562nm, and calculate the total cell protein concentration.

[0104] 6. Western blotting (WB) detection of target protein expression: Total protein (10 μg) was separated by electrophoresis (200 V, 22 min) using precast protein electrophoresis gel Bolt™ 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel (Invitrogen, NW04120BOX). The separated proteins on the gel were transferred to iBlot 2 Transfer Stacks, PVDF (Invitrogen, IB24001) membrane under the action of gradient voltage (20 V, 1 min; 23 V, 4 min; 25 V, 2 min), and then incubated in 1× TBST containing 5% skim milk powder at room temperature, 20 r / min, and blocked for 1 h.

[0105] The primary antibody used monoclonal antibody diluent, Anti-Monkeypox Virus / MPXV M1R Antibdy (Pujian Bio, SAA0283), Anti-Monkeypox Virus / MPXV B6R / SL-159 Polyclonal Antibody (Pujian Bio, PVV13501), Anti-MPXV-A29L rRmAb (Vazyme, RM3387), Anti-MPXV-E8L rRmAb (Vazyme, RM3391), Anti-MPXV-A35R rRmAb (Vazyme, RM3395), 20r / min, incubated at room temperature for 2h. Wash the membrane with 1×TBST, 60r / min, incubated at room temperature for 10min, and repeat 3 times to completely remove the residual primary antibody. The secondary antibody was horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody diluent (1:2000), HRP Goat anti-Mouse IgG (H+L) (Abclone, AS003) or horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody diluent (1:1000), (Biyuntian, A0208), 20r / min, incubated at room temperature for 1h. The membrane was washed with 1×TBST, 60r / min, incubated at room temperature for 10min, and repeated 3 times to completely remove the residual secondary antibody.

[0106] The membrane was incubated with the ECL chemiluminescent ultrasensitive colorimetric kit (Yisheng Bio, 36208ES60) at room temperature in the dark for 3 minutes, and the HRP-labeled antibody-bound antigen was detected in a chemiluminometer. The bands were exposed and compared with the protein marker, PageRuler™ Prestained Protein Ladder (Invitrogen, 26617) bands to verify the expression of the target antigen protein. The membrane regeneration solution (Solebo, SW3020) was used to remove the antibodies on the membrane, and the membrane was blocked again, and then the internal reference antibody was incubated to detect the consistency of the protein loading amount. The primary antibody used β-actin rabbit monoclonal antibody dilution (1:50000), ACTBRabbit mAb (Abclonal, AC038), and the secondary antibody used horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody dilution (1:10000), Goat Anti-Rabbit IgG Secondary Antibody (HRP) (Sino Biological, SSA004).

[0107] According to the above method, transfection and detection of mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000A, mRNA-2949B and mRNA-2949C were performed respectively. The results are as follows: Figure 3 The results showed that after the in vitro synthesized monkeypox virus mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000A, mRNA-2949B and mRNA-2949C were transfected into HEK293T cells, the highly expressed monkeypox virus A29L, M1R, E8L, A35R and B6R target antigens were detected by WB.

[0108] 4. Preparation of multi-antigen monkeypox mRNA vaccine

[0109] (1) Preparation of mRNA-2949-LNP, mRNA-3000-LNP, mRNA-2949A-LNP, mRNA-3000A-LNP, mRNA-2949B-LNP and mRNA-2949C-LNP lipid nanoparticle vaccines

[0110] Cationic lipid (SM102), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and DMG-PEG2000 were completely dissolved in ethanol. The above lipid ethanol solution was mixed at a molar ratio of 50:10:38.5:1.5, and mixed with 20mM sodium citrate buffer (pH 4.0) of mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000A, mRNA-2949B, and mRNA-2949C at a volume ratio of 1:3 (lipid: mRNA) at a flow rate of 12mL / min in the Myanna Nanodrug Preparation System. The collected sample solution was diluted 50 times in DPBS buffer and then concentrated by centrifugation through a 50kDa PES ultrafiltration tube at 4°C and 2000×g to remove the ethanol content in the sample solution. Finally, the vaccine preparation that passed through the 0.22 μm filter membrane was adjusted to a suitable concentration with DPBS buffer to continue the experiment.

[0111] Example 2. Detection of serum antibodies in mice immunized with monkeypox virus mRNA vaccine

[0112] 1. Muscle immunization of mice with multi-antigen monkeypox virus mRNA vaccine:

[0113] The experimental animals BALB / c mice (female, 6-8 weeks, 16-18g, Beijing Weitong Lihua) were randomly divided into monkeypox virus mRNA-2949-LNP (mRNA=25µg / mouse), mRNA-3000-LNP (mRNA=25µg / mouse), mRNA-2949A-LNP (mRNA=25µg / mouse), mRNA-3000A-LNP (mRNA=25µg / mouse), mRNA-2949B-LNP (mRNA=25µg / mouse), mRNA-2949C-LNP (mRNA=25µg / mouse) and negative control group (5 mice in each group, normally fed), and the mice were immunized by intramuscular injection. Immunization was performed twice, and sufficient mouse serum was obtained by orbital bleeding on the 10th day after immunization.

[0114] II. Mouse muscle immunization and serum antibody detection of multi-antigen monkeypox virus mRNA vaccine

[0115] The experimental animals BALB / c mice (female, 6-8 weeks, 16-18g, Beijing Weitong Lihua) were randomly divided into different immunization groups of mRNA-2949-LNP (mRNA=25µg / mouse), mRNA-3000-LNP (mRNA=25µg / mouse), mRNA-2949A-LNP (mRNA=25µg / mouse), mRNA-3000A-LNP (mRNA=25µg / mouse), mRNA-2949B-LNP (mRNA=25µg / mouse), mRNA-2949C-LNP (mRNA=25µg / mouse) and negative control group (PBS, pH=7.4) (5 mice in each group, normal feeding), and the mice were immunized by intramuscular injection. The interval between the two immunizations of the LNP-mRNA group was 14 days. On the 10th day after each immunization, orbital blood was collected to obtain sufficient mouse serum. The ELISA method was used to detect the production of monkeypox virus antigen-specific binding antibody IgG in mouse immune serum, thereby evaluating the in vivo immune efficacy of the multi-cancer monkeypox virus vaccine.

[0116] The ELISA method was used to detect the production of monkeypox virus antigen-specific binding antibody IgG in the serum of immunized mice. The specific detection method is as follows:

[0117] (1) Coating: Dilute A29L, A35R, B6R, M1R, and E8L proteins to 1 ng / µl using carbonate buffer (50 mM, pH 9.6, filtered with a 0.22 μm filter membrane). Add 100 μL of protein dilution to each well of a 96-well plate, seal, and incubate at 4°C overnight.

[0118] (2) Washing: After overnight coating, pour the 96-well plate to remove the protein coating solution, add 200 μL of washing solution (1×TBS containing 0.2% Tween-20) to each well, shake gently by hand for 30 seconds, and pat dry on paper. Repeat 6 times;

[0119] (3) Blocking: Add 200 μl of blocking solution (1×TBS containing 2% BSA) to each well and incubate at 37°C for 2 h.

[0120] (4) Washing: Pour the 96-well plate to remove the blocking solution, add 200 μl of washing solution (1× TBS containing 0.2% Tween-20) to each well, shake gently by hand for 30 seconds, and pat dry on paper. Repeat 6 times;

[0121] (5) Incubation with primary antibody: Immunized mouse serum was diluted 10-fold with antibody diluent (washing solution containing 0.5% BSA) to obtain serum with different dilutions from 10^-1 to 10^-6. 100 μl of serum diluent was added to each well and incubated at 37°C for 2 h.

[0122] (6) Washing: Pour the 96-well plate to remove the serum diluent, add 200 μL of washing solution (1× TBS containing 0.2% Tween-20) to each well, shake gently by hand for 30 seconds, and pat dry on paper. Repeat 6 times;

[0123] (7) Incubation with secondary antibody: dilute HRP-conjugated Goat anti-Mouse IgG (H+L) (ABclonal, AS003) 5000 times with antibody diluent (washing buffer containing 0.5% BSA) to obtain secondary antibody diluent. Add 100 μl of secondary antibody diluent to each well and incubate at 37°C for 1 h.

[0124] (8) Washing: Pour the 96-well plate to remove the secondary antibody dilution solution, add 200 μl of washing solution (1× TBS containing 0.2% Tween-20) to each well, shake gently by hand for 30 seconds, and pat dry on paper. Repeat 6 times;

[0125] (9) Color development: Add 100 μl TMB substrate (Tian Gen, RA107) to each well and incubate for 20 min at room temperature in the dark.

[0126] (10) Stop color development: Add 50 μL of 2 M HO to each well. 2 SO 4 , the OD value of A450 was detected on an ELISA reader;

[0127] (11) Determination of serum binding antibody IgG titer: If the OD of a certain dilution of serum / the OD of the negative control is ≥ 2.1, and the OD of the next dilution factor / the OD of the negative control is < 2.1, the dilution factor is the antibody titer corresponding to the serum sample (if the OD of the negative control is < 0.05, it is calculated as 0.05).

[0128] The results are as follows Figure 4It was shown that high titer antigen-specific binding antibody IgG was detected in the sera of mice immunized with monkeypox virus mRNA vaccines mRNA-2949-LNP, mRNA-3000-LNP, mRNA-2949A-LNP, mRNA-3000A-LNP, mRNA-2949B-LNP and mRNA-2949C-LNP. The average binding antibody IgG titer of A29L was approximately 1:12800 (mRNA-2949-LNP group), 1:10400 (mRNA-3000-LNP group), 1:26000 (mRNA-2949A-LNP group), and 1:18000 (mRNA-3000A-LNP group). The average binding antibody IgG titer of A35R was approximately 1:14000 (mRNA-2949-LNP group), 1:44000 (mRNA-3000-LNP group), 1:28000 (mRNA-2949A-LNP group), 1:28000 (mRNA-3000A-LNP group), 1:10200 (mRNA-2949B-LNP group) and 1:120000 (mRNA-2949C-LNP group); the average binding antibody IgG titer of M1R was approximately 1:64000 (mRNA-2949-LNP group), 1:44000 (mRNA-3000-LNP group), 1:28000 (mRNA-2949A-LNP group), 1:28000 (mRNA-3000A-LNP group), 1:10200 (mRNA-2949B-LNP group) and 1:120000 (mRNA-2949C-LNP group). The average titers of B6R binding antibody IgG were approximately 1:14000 (mRNA-2949-LNP group), 1:140000 (mRNA-3000-LNP group), 1:660000 (mRNA-2949A-LNP group), 1:240000 (mRNA-3000A-LNP group), 1:280000 (mRNA-2949B-LNP group) and 1:520000 (mRNA-2949C-LNP group). 0 (mRNA-3000A-LNP group), 1:84000 (mRNA-2949B-LNP group), and 1:140000 (mRNA-2949C-LNP group); the average E8L binding antibody IgG titer was approximately 1:320000 (mRNA-2949-LNP group), 1:600000 (mRNA-3000-LNP), 1:1160000 (mRNA-2949A-LNP group), 1:960000 (mRNA-3000A-LNP group), 1:1000000 (mRNA-2949B-LNP group), and 1:180000 (mRNA-2949C-LNP group) (e.g. Figure 4 as shown).

[0129] 3. Detection of neutralizing antibodies against live vaccinia virus of Tiantan strain in serum of mice immunized with monkeypox virus mRNA vaccine

[0130] Evaluation of the neutralization effect of mouse immune serum on live poxvirus by using live vaccinia Tiantan strain virus and determination of neutralizing antibody titer NT 50 , thereby evaluating the in vivo immune efficacy of monkeypox virus mRNA-2949-LNP, mRNA-3000-LNP, mRNA-2949A-LNP, mRNA-3000A-LNP, mRNA-2949B-LNP and mRNA-2949C-LNP vaccines.

[0131] Vaccinia virus neutralizing antibody NT 50 Potency determination:

[0132] The live virus of the vaccinia strain of Tiantan was used to evaluate the neutralizing effect of antibodies in the serum of mice immunized with the monkeypox virus mRNA vaccine. The live virus of the vaccinia strain of Tiantan used in the evaluation was provided by the China Food and Drug Inspection Institute. The specific detection method is as follows:

[0133] Mouse immune serum was diluted three times from 1 / 30 in DMEM complete medium to obtain 6 different serum dilutions, which were incubated with the virus at 37°C. A virus-free cell control group and a serum-free virus control group were also set up. After incubation for 1 hour, 2×10 4 Vero cells, 37°C, 5% CO 2 Cell culture was performed under the condition of 48 hours. Since the live virus of Tiantan strain enters the cell, it will express firefly luciferase. After 48 hours, it will react with the luminescent substrate and perform luminescence detection. By comparing with the luminescence value of the virus control group, the percentage of virus inhibition is calculated. The calculation formula can be used to calculate the dilution multiple of the serum when the pseudovirus is 50% inhibited, so as to calculate the half inhibition dilution. The half neutralization dilution NT is expressed by the half inhibition dilution. 50 , that is, the neutralizing activity of serum antibodies against the virus.

[0134] The results are as follows Figure 5 The results showed that the sera of mice immunized with monkeypox virus mRNA-2949-LNP, mRNA-3000-LNP, mRNA-2949A-LNP, mRNA-3000A-LNP, mRNA-2949B-LNP and mRNA-2949C-LNP groups all had neutralizing effects on the live vaccinia Tiantan strain virus. After two immunizations, the average virus neutralizing antibody titer NT 50They are mRNA-2949: ~1:2323, mRNA-3000: ~1:2185, mRNA-2949A: ~1:5938, mRNA-3000A: ~1:5042, mRNA-2949B: ~1:5053 and mRNA-2949C: ~1:6163.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An mRNA, characterized in that The mRNA is shown in any one of A1) to A2) below: A1) mRNA sequence obtained by replacing T in SEQ ID No. 6 in the sequence listing with U; A2) A sequence in which a Cap structure is connected to the 5' end of the mRNA in A1) and / or a ployA structure is connected to the 3' end.

2. Use of the mRNA according to claim 1 in the preparation of an mRNA vaccine for preventing monkeypox virus infection.

3. An mRNA vaccine, characterized in that: The active ingredient of the vaccine is the mRNA according to claim 1.

4. A biological material related to the mRNA according to claim 1, characterized in that: The biological material is any one of B1) to B6): B1) a nucleic acid molecule encoding the mRNA according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant plasmid containing the nucleic acid molecule described in B1), or a recombinant plasmid containing the expression cassette described in B2); B5) A recombinant bacterium containing the nucleic acid molecule described in B1), or a recombinant bacterium containing the expression cassette described in B2), or a recombinant bacterium containing the recombinant vector described in B3).

5. The biomaterial according to claim 4, characterized in that The B3) recombinant vector is a sequence in which the nucleic acid molecule sequence encoding the mRNA according to claim 1 replaces the sequence between the vector T7 promoter sequence and the BsaI site of the vector pVAX, while keeping the other sequences of the vector unchanged.

6. An mRNA complex comprising a delivery vector and the mRNA of claim 1, wherein the delivery vector comprises any one of an ionizable liposome, an ionizable protein, an ionizable polymer and an ionizable micelle.

7. An mRNA complex comprising a delivery vector and the mRNA of claim 1, wherein the delivery vector comprises an ionizable lipid nanoparticle.

8. The mRNA complex according to claim 6, characterized in that The ionizable liposome is a cationic liposome, the ionizable protein is a cationic protein, the ionizable polymer is a cationic polymer, and the ionizable micelle is a cationic micelle.

9. The mRNA complex according to claim 7, characterized in that The ionizable lipid nanoparticles are cationic lipid nanoparticles.

10. The mRNA complex according to claim 7, characterized in that The mRNA complex is an ionizable lipid-mRNA lipid nanoparticle, which comprises an ionizable lipid, the mRNA according to claim 1, a PEGylated lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol.

11. Use of the biological material according to any one of claims 4 to 5 or the mRNA complex according to any one of claims 6 to 10 in the preparation of an mRNA vaccine for preventing monkeypox virus.

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