Rnas, combinations of rnas and uses thereof, multivalent monkeypox vaccines

Abstract

The application provides an RNA, an RNA combination and application thereof, and a multivalent monkeypox vaccine, and relates to the technical field of biotechnology.The mRNA provided by the application is obtained after codon optimization, and can express antigen proteins in vitro cells with high efficiency; the mRNA combination provided by the application has high expression, and after immunizing mice, high-titer neutralizing antibodies against the live virus of the vaccinia virus Tianlu strain can be detected, and the mRNA combination has good immunogenicity, and is of great significance for the prevention of the monkeypox virus.The multivalent monkeypox mRNA vaccine provided by the application can realize stable and safe expression in vivo and effectively activate an immune response, and can cause a neutralizing antibody response, and in the serum of the immunized mice, high-titer neutralizing antibodies against the live virus of the vaccinia virus Tianlu strain can be detected.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an RNA, RNA combinations and their applications, and a multivalent monkeypox vaccine. Background Technology

[0002] Globally disseminating safe and effective vaccines is the most powerful weapon against monkeypox and key to ending the monkeypox epidemic. Currently, there is no specific vaccine for monkeypox. Monkeypox, along with smallpox and cowpox, belongs to the orthopoxvirus family. Smallpox vaccines offer cross-protection; vaccination against smallpox has an 85% effectiveness rate in preventing monkeypox or reducing the severity of infection. Currently, only two vaccines have been approved by the FDA for the prevention of monkeypox: 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 an effective and safe next-generation monkeypox-specific vaccine. Furthermore, monkeypox continues to ravage the world, and the public health crisis it causes remains unresolved.

[0003] Monkeypox virus is an enveloped double-stranded DNA virus with two distinct genetic evolutionary branches—the Central African branch and the West African branch. The West African branch has a case fatality rate of approximately 3.6%; the Central African branch has historically caused more severe disease, with a case fatality rate of approximately 10.6%, and is considered more infectious. In this current monkeypox outbreak, all cases reported outside of Africa, including those currently spreading, are caused by the West African branch. Since the eradication of smallpox in 1980 and the subsequent cessation of smallpox vaccination, monkeypox has become the most important orpox virus in public health. Reports also indicate that only 9% of patients infected in the current outbreak had previously been vaccinated against smallpox.

[0004] mRNA vaccines offer technological advantages such as shorter development cycles and faster production capacity, making them more suitable for the emergency development and production of preventative vaccines against monkeypox virus. However, the design of mRNA vaccines for monkeypox virus is challenging, especially since achieving complete and efficient viral expression is affected by various factors.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide an mRNA with a high expression level.

[0007] The second objective of this invention is to provide an mRNA combination that can express antigen proteins in in vitro cells and can produce high-titer neutralizing antibodies against the Tian Tan strain of live vaccinia virus after immunizing mice. It has good immunogenicity and is of great significance for the prevention of monkeypox virus.

[0008] A third objective of this invention is to provide a biomaterial.

[0009] The fourth objective of this invention is to provide an application of mRNA combination in the preparation of monkeypox vaccine.

[0010] The fifth objective of this invention is to provide an mRNA complex.

[0011] The sixth objective of this invention is to provide a multivalent monkeypox mRNA vaccine.

[0012] A seventh objective of this invention is to provide a pharmaceutical composition.

[0013] The eighth objective of this invention is to provide a reagent kit.

[0014] In a first aspect, the present invention provides an mRNA, wherein the mRNA comprises, from 5' to 3', a 5'UTR, a Kozak sequence, a coding region, a 3'UTR, and a Poly A tail;

[0015] The nucleic acid sequence of the 5'UTR is as shown in SEQ ID NO.1, or a sequence that has more than 95% homology with SEQ ID NO.1;

[0016] The nucleic acid sequence of the 3'UTR is as shown in SEQ ID NO.2, or a sequence that has more than 95% homology with SEQ ID NO.2;

[0017] The nucleic acid sequence of the coding region is as shown in any one of SEQ ID NO.4 to SEQ ID NO.9, or a sequence that has more than 95% homology with any one of SEQ ID NO.4 to SEQ ID NO.9.

[0018] As a further technical solution, the Kozak sequence includes GCC, ACC, GCCACC, or GCCANN;

[0019] Preferably, the nucleic acid sequence with the Poly A tail is as shown in SEQ ID NO.3, or is a sequence with more than 95% homology to SEQ ID NO.3.

[0020] In a second aspect, the present invention provides an mRNA combination comprising: mRNA encoding monkeypox virus A29L, mRNA encoding monkeypox virus A35R, mRNA encoding monkeypox virus M1R, and mRNA encoding monkeypox virus B6R.

[0021] The mRNA sequence encoding monkeypox virus A29L is shown in SEQ ID NO.10, or a sequence having more than 95% homology with SEQ ID NO.10;

[0022] The mRNA sequence encoding monkeypox virus A35R is shown in SEQ ID NO.11 or SEQ ID NO.12, or a sequence having more than 95% homology with SEQ ID NO.11 or SEQ ID NO.12;

[0023] The mRNA sequence encoding monkeypox virus M1R is shown in SEQ ID NO.13, or a sequence having more than 95% homology with SEQ ID NO.13;

[0024] The mRNA sequence encoding monkeypox virus B6R is shown in SEQ ID NO.14 or SEQ ID NO.15, or a sequence having more than 95% homology with SEQ ID NO.14 or SEQ ID NO.15.

[0025] Thirdly, the present invention provides biological materials comprising at least one of the following B1) to B6):

[0026] B1) Transcribe the nucleic acid molecule of the mRNA, or transcribe the nucleic acid molecule of the mRNA combination;

[0027] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0028] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0029] B4) A recombinant plasmid containing the nucleic acid molecule described in B1), or a recombinant plasmid containing the expression cassette described in B2);

[0030] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0031] 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).

[0032] Fourthly, the present invention provides the application of the above-mentioned mRNA or mRNA combination in the preparation of monkeypox vaccine.

[0033] Fifthly, the present invention provides an mRNA complex comprising a delivery vector and the mRNA or a combination of the mRNAs.

[0034] As a further technical solution, the delivery carrier includes any one of ionizable liposomes, cationic liposomes, ionizable proteins, cationic proteins, ionizable polymers, cationic polymers, ionizable micelles, cationic micelles, ionizable lipid nanoparticles, or cationic lipid nanoparticles.

[0035] Preferably, the delivery carrier is YK009;

[0036] Preferably, the mRNA complex further includes 1,2-distearate-sn-glycerol-3-phosphate choline, cholesterol, and DMG-PEG2000.

[0037] In a sixth aspect, the present invention provides a valence monkeypox mRNA vaccine, comprising the mRNA, the mRNA combination, or the mRNA complex.

[0038] Preferably, in the mRNA combination, the sequence of the mRNA encoding monkeypox virus A35R is shown in SEQ ID NO.11, and the sequence of the mRNA encoding monkeypox virus B6R is shown in SEQ ID NO.14;

[0039] Alternatively, the sequence of mRNA encoding monkeypox virus A35R is shown in SEQ ID NO.12; the sequence of mRNA encoding monkeypox virus B6R is shown in SEQ ID NO.15.

[0040] In a seventh aspect, the present invention provides a pharmaceutical composition comprising at least one of the mRNA, the mRNA combination, the biological material, the mRNA complex, or the multivalent monkeypox mRNA vaccine, and optionally a pharmaceutical carrier.

[0041] Eighthly, the present invention provides a kit comprising at least one of the aforementioned mRNA, the aforementioned mRNA combination, the aforementioned biological material, the aforementioned mRNA complex, or the aforementioned multivalent monkeypox mRNA vaccine.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The successful development of mRNA vaccines largely depends on the optimization of the mRNA sequence itself. The mRNA provided in this invention, as the active ingredient in the vaccine, is obtained after codon optimization and can efficiently express the antigen protein in both in vitro and cellular environments. The mRNA combination provided in this invention exhibits high expression levels and, after immunizing mice, produces high-titer neutralizing antibodies against the Tian Tan strain of live vaccinia virus, demonstrating good immunogenicity and significant importance for the prevention of monkeypox virus.

[0044] The multivalent monkeypox mRNA vaccine provided by this invention can achieve stable and safe expression and effectively activate the immune response in vivo, and can induce a neutralizing antibody response. High-titer neutralizing antibodies against the Tian Tan strain of live vaccinia virus can be detected in the serum of immunized mice. Attached Figure Description

[0045] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0046] Figure 1 This is a schematic diagram of the structures of the active components of the monkeypox virus mRNA vaccine provided by the present invention: mRNA-M351, mRNA-M564, mRNA-M630, mRNA-M837, mNRA-M972 and mRNA-M1038.

[0047] Figure 2 This is a graph showing the quality analysis results in Embodiment 1 of the present invention;

[0048] Figure 3 This is a diagram showing the cell transfection protein expression detection results in Example 1 of the present invention;

[0049] Figure 4 This refers to the detection of specific antibody IgG in the serum of mice after immunization in Example 2 of the present invention.

[0050] Figure 5 The neutralizing antibody NT against live vaccinia Tiantan strain vaccinia virus in the serum of immunized mice in Example 2 of this invention. 50 The image shows the test results. Detailed Implementation

[0051] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0052] The term "neutralizing antibody" generally refers to the antibody produced when microorganisms invade the human body. While many types of antibodies are produced, only a portion of these antibodies can quickly recognize the microorganisms and "capture" them before they invade human cells, protecting the body from infection. This process is called neutralization, and the antibodies that play this role are called neutralizing antibodies.

[0053] The term "adjuvant" refers to a reagent that increases, stimulates, activates, enhances, or modulates the immune response against the active ingredient of a composition at the cellular or humoral level.

[0054] In a first aspect, the present invention provides an mRNA, wherein the mRNA comprises, from 5' to 3', a 5'UTR, a Kozak sequence, a coding region, a 3'UTR, and a Poly A tail;

[0055] The nucleic acid sequence of the 5'UTR is as shown in SEQ ID NO.1, or a sequence that has more than 95% homology with SEQ ID NO.1;

[0056] The nucleic acid sequence of the 3'UTR is as shown in SEQ ID NO.2, or a sequence that has more than 95% homology with SEQ ID NO.2;

[0057] The nucleic acid sequence of the coding region is as shown in any one of SEQ ID NO.4 to SEQ ID NO.9, or a sequence that has more than 95% homology with any one of SEQ ID NO.4 to SEQ ID NO.9.

[0058] It should be noted that the terms "homology" and "identity" used in this invention are synonymous, referring to a similarity (including but not limited to) of 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%, and 99.9% in terms of amino acid or nucleotide sequence compared to the sequences disclosed in this invention, while still retaining the same function as the original amino acid or nucleotide sequence. In this invention, 5'UTR refers to the 5'UTR excluding the Kozak sequence.

[0059] The mRNA provided by this invention as an active ingredient in vaccines is obtained after codon optimization, and it can efficiently express antigen proteins in in vitro cells.

[0060] In some preferred embodiments, the Kozak sequence includes GCC, ACC, GCCACC, or GCCANN;

[0061] Preferably, the nucleic acid sequence with the Poly A tail is as shown in SEQ ID NO.3, or is a sequence with more than 95% homology to SEQ ID NO.3.

[0062] In a second aspect, the present invention provides an mRNA combination comprising: mRNA encoding monkeypox virus A29L, mRNA encoding monkeypox virus A35R, mRNA encoding monkeypox virus M1R, and mRNA encoding monkeypox virus B6R.

[0063] The mRNA sequence encoding monkeypox virus A29L is shown in SEQ ID NO.10, or a sequence having more than 95% homology with SEQ ID NO.10;

[0064] The mRNA sequence encoding monkeypox virus A35R is shown in SEQ ID NO.11 or SEQ ID NO.12, or a sequence having more than 95% homology with SEQ ID NO.11 or SEQ ID NO.12;

[0065] The mRNA sequence encoding monkeypox virus M1R is shown in SEQ ID NO.13, or a sequence having more than 95% homology with SEQ ID NO.13;

[0066] The mRNA sequence encoding monkeypox virus B6R is shown in SEQ ID NO.14 or SEQ ID NO.15, or a sequence having more than 95% homology with SEQ ID NO.14 or SEQ ID NO.15.

[0067] In this invention, monkeypox (MPX), an infectious disease, is a viral zoonotic disease caused by monkeypox virus (MPXV). Intracellular mature viruses (IMVs) and extracellular enveloped viruses (EEVs) express different viral proteins. A29L and M1R are surface proteins of mature virus particles, while A35R and B6R are surface proteins of enveloped virus particles. The A29L protein is a monoclonal antibody target for MPXV (monkeypox virus), mediating recognition of host cells and catalytic fusion between the virus and host cells. A35R is an important component of enveloped virus particles and also a potential serological detection target. The B6R protein is present on the membrane of EEV particles, negatively regulating complement activation, and its interaction with A33 or A34 determines its intracellular localization. The combination of intracellular mature virus particles and extracellular enveloped virus particles provides better protective efficacy.

[0068] The mRNA combination provided by this invention as a vaccine active ingredient, obtained after codon optimization, can efficiently express antigen proteins in in vitro cells, and can detect high-titer neutralizing antibodies against the Tian Tan strain of live vaccinia virus after immunizing mice, demonstrating good immunogenicity and significant importance for the prevention of monkeypox virus.

[0069] Thirdly, the present invention provides biological materials comprising at least one of the following B1) to B6):

[0070] B1) Transcribe the nucleic acid molecule of the mRNA, or transcribe the nucleic acid molecule of the mRNA combination;

[0071] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0072] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0073] B4) A recombinant plasmid containing the nucleic acid molecule described in B1), or a recombinant plasmid containing the expression cassette described in B2);

[0074] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0075] 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).

[0076] In this invention, the coding sequence of a protein can be cloned into a plasmid using genetic engineering techniques, followed by in vitro transcription for mRNA synthesis. Preferably, the process includes: 1) cloning the DNA fragment corresponding to the mRNA into an expression plasmid to obtain a recombinant plasmid; 2) transforming the recombinant plasmid into host cells to obtain recombinant cells, extracting the plasmid from the amplified recombinant cells, and performing PCR amplification to obtain a DNA template for in vitro expression of mRNA; 3) constructing an in vitro RNA synthesis system including the DNA template to synthesize the active ingredient mRNA in vitro. In this invention, the specific sequence of the DNA fragment can be determined according to the base pairing principle.

[0077] In one implementation, after in vitro transcription, the transcribed RNA product is capped, and the resulting mRNA has a cap (Cap-1) structure attached to its 5' end.

[0078] In one embodiment, the recombinant vector is vector PVAX1. Specifically, a DNA template of the mRNA sequence is constructed between the last base of the T7 promoter of vector pVAX1—the multiple cloning site XbaI—to obtain a recombinant plasmid. In vitro transcription is initiated using the T7 promoter sequence on the vector under the action of T7 transcriptase.

[0079] 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.

[0080] Fourthly, the present invention provides the application of the above-mentioned mRNA or mRNA combination in the preparation of monkeypox vaccine.

[0081] The mRNA provided by this invention has high expression level and good immunogenicity, and can be used in the preparation of monkeypox vaccine.

[0082] Fifthly, the present invention provides an mRNA complex comprising a delivery vector and the mRNA or a combination of the mRNAs.

[0083] In some preferred embodiments, the delivery carrier comprises any one of ionizable lipids (MC3, SM102, ALC0315, Lipid 5, DOTAP), cationic lipids, ionizable proteins, cationic proteins, ionizable polymers, cationic polymers, ionizable micelles, cationic micelles, ionizable lipid nanoparticles, or cationic lipid nanoparticles, preferably ionizable lipid nanoparticles (LPN), more preferably YK009 (2-octyldecyl 6-((4-(decoxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate).

[0084] In one embodiment, the mRNA complex is selected from ionizable lipid-mRNA complexes, cationic lipid-mRNA complexes, or novel cationic lipid-mRNA complexes, ionizable lipid-mRNA lipid nanoparticles, cationic lipid-mRNA lipid nanoparticles, or novel cationic lipid-mRNA lipid nanoparticles. It should be noted that ionizable lipid-mRNA complexes refer to a mixture of ionizable lipids and the mRNA of the present invention; the same applies to cationic lipid-mRNA complexes and novel cationic lipid-mRNA complexes. Ionizable lipid-mRNA lipid nanoparticles refer to lipid nanoparticles formed by ionizable lipids, 1,2-distearate-sn-glycerol-3-phosphocholine, cholesterol, and DMG-PEG2000 together with the mRNA of the present invention; the same applies to cationic lipid-mRNA lipid nanoparticles or novel cationic lipid-mRNA lipid nanoparticles.

[0085] Preferably, the ionizable lipid-mRNA complex, cationic lipid-mRNA complex, or novel cationic lipid-mRNA complex further comprises protamine, PEGylated lipids, 1,2-diole-sn-glycerol-3-phosphate ethanolammonium, and cholesterol.

[0086] Preferably, the ionizable lipid-mRNA complex nanoparticles, cationic lipid-mRNA nanoparticles, or novel cationic lipid-mRNA nanoparticles further include PEGylated lipids, 1,2-distearate-sn-glycerol-3-phosphocholine, and cholesterol.

[0087] In one embodiment, the mRNA complex includes lipid nanoparticles LNP-mRNA prepared by combining mRNA with ionizable lipids, and novel cationic lipid material-mRNA lipid nanoparticles prepared by combining mRNA with ionizable lipid YK009.

[0088] In one embodiment, the method for preparing the mRNA complex includes: mixing the mRNA with an ionizable lipid material and then packaging it with lipids; wherein the ionizable lipid material may be MC3, SM102, ALC0315, Lipid 5, DOTAP, etc.

[0089] In a preferred embodiment, the method for preparing the mRNA complex includes dissolving and mixing an ionizable lipid material with 1,2-distearate-sn-glycerol-3-phosphate choline and DMG-PEG2000, and then mixing it with mRNA.

[0090] In a sixth aspect, the present invention provides a valence monkeypox mRNA vaccine, comprising the mRNA, the mRNA combination, or the mRNA complex.

[0091] Through numerous experiments, the inventors ultimately discovered that a specific combination of the framework sequence and coding sequence of this invention enables the prepared vaccine to achieve better immunogenicity and stability.

[0092] The multivalent monkeypox virus mRNA vaccine provided by this invention is a vaccine against monkeypox virus, and its administration targets 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 used to prevent monkeypox virus infection. The term "prevention" in this invention refers to all behaviors that avoid symptoms or delay the onset of specific symptom stress by administering the product of this invention before or after the onset of disease development.

[0093] Preferably, in the mRNA combination, the sequence of the mRNA encoding monkeypox virus A35R is shown in SEQ ID NO.11, and the sequence of the mRNA encoding monkeypox virus B6R is shown in SEQ ID NO.14;

[0094] Alternatively, the sequence of mRNA encoding monkeypox virus A35R is shown in SEQ ID NO.12; the sequence of mRNA encoding monkeypox virus B6R is shown in SEQ ID NO.15.

[0095] In a preferred embodiment, the valvular monkeypox mRNA vaccine further includes an adjuvant.

[0096] In a seventh aspect, the present invention provides a pharmaceutical composition comprising at least one of the said mRNA, the said mRNA combination, the said biological material, the said mRNA complex, or the said multivalent monkeypox mRNA vaccine, and optionally a pharmaceutical carrier. The present invention provides the use of a product comprising the said mRNA, the said mRNA combination, the said biological material, the said mRNA complex, or the said multivalent monkeypox mRNA vaccine in the preparation of a medicament for the prevention and / or treatment of monkeypox virus infection.

[0097] Eighthly, the present invention provides a kit comprising at least one of the mRNA, the mRNA combination, the biological material, the mRNA complex, or the multivalent monkeypox mRNA vaccine.

[0098] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0099] Example 1: Sequence design, preparation, and in vitro cell antigen expression detection of monkeypox virus mRNA vaccine

[0100] I. Sequence Design of Monkeypox mRNA Vaccine

[0101] The sequence design of the monkeypox mRNA vaccine employs an optimized mRNA backbone code sequence to enhance mRNA stability and protein expression efficacy. The CDS of the monkeypox virus mRNA is composed of optimized codons, determining the amino acid sequences of the protective immunogenic proteins of monkeypox virus IMV and EEV.

[0102] To achieve in vitro transcription of mRNA, the monkeypox virus mRNA vaccine sequence template was constructed between the last base of the T7 promoter on the vector PVAX1 and the multiple cloning site XbaI. In vitro transcription was initiated by the T7 promoter sequence under the action of T7 transcriptase.

[0103] Therefore, the active ingredients of monkeypox mRNA vaccine, mRNA-M351, mRNA-M564, mRNA-M630, mRNA-M837, mNRA-M972, and mRNA-M1038, were designed and optimized. Figure 1 As shown: mRNA-M351 ( Figure 1 As shown in Figure 1), mRNA-M564 ( Figure 1 As shown in Figure 2), mRNA-M630 ( Figure 1 As shown in Figure 3), mRNA-M837 ( Figure 1 As shown in Figure 4), mRNA-M972 ( Figure 1 As shown in Figure 5), and mRNA-M1038 ( Figure 1 The sequences shown in (see SEQ ID NO. 10 to SEQ ID NO. 15) are as shown in SEQ ID NO. 6.

[0104] mRNA-M351 DNA template sequence (SEQ ID No. 10):

[0105] From the 5' end, positions 1 to 52 are 5'-UTR, positions 49-52 are Kozak sequences. Those skilled in the art can select and replace Kozak sequences different from those of the present invention using conventional techniques. For example, the Kozak sequence can be replaced with GCC, ACC, GCCACC, or GCCANN. Positions 53 to 385 are CDS-A29L (encoding amino acids 1-110 of the monkeypox virus A29L protein), positions 386 to 511 are 3'-UTR, and positions 512 to 631 are Poly A tails.

[0106] mRNA-M564 sequence (SEQ ID No. 11):

[0107] From the 5' end, positions 1 to 52 are 5'-UTR, and positions 49-52 are Kozak sequences. Those skilled in the art can select and replace Kozak sequences that are different from those of this invention using conventional techniques. For example, the Kozak sequence can be replaced with GCC, ACC, GCCACC, or GCCANN. Positions 53 to 598 are CDS-A29L (encoding amino acids 1-181 of the monkeypox virus A35R protein), positions 599 to 724 are 3'-UTR, and positions 725 to 844 are Poly A tails.

[0108] mRNA-M630 sequence (SEQ ID No.12):

[0109] From the 5' end, positions 1 to 52 are 5'-UTR, and positions 49-52 are Kozak sequences. Those skilled in the art can select and replace Kozak sequences that are different from those of this invention using conventional techniques. For example, the Kozak sequence can be replaced with GCC, ACC, GCCACC, or GCCANN. Positions 53 to 664 are CDS-A35R (positions 53-121 encode the tPA signal peptide, and positions 122-664 encode amino acids 1-181 of the monkeypox virus A35R protein). Positions 665 to 790 are 3'-UTR, and positions 791 to 910 are Poly A tails.

[0110] mRNA-M837 sequence (SEQ ID No.13):

[0111] From the 5' end, positions 1 to 52 are 5'-UTR, and positions 49-52 are Kozak sequences. Those skilled in the art can select and replace Kozak sequences that are different from those of this invention using conventional techniques. For example, the Kozak sequence can be replaced with GCC, ACC, GCCACC, or GCCANN. Positions 53 to 871 are CDS-M1R (positions 53-121 encode the tPA signal peptide, and positions 122-871 encode amino acids 1-249 of the monkeypox virus M1R protein). Positions 872 to 997 are 3'-UTR, and positions 998 to 1117 are Poly A tails.

[0112] mRNA-M972 sequence (SEQ ID No.14):

[0113] From the 5' end, positions 1 to 52 are 5'-UTR, and positions 49-52 are Kozak sequences. Those skilled in the art can select and replace Kozak sequences that are different from those of this invention using conventional techniques. For example, the Kozak sequence can be replaced with GCC, ACC, GCCACC, or GCCANN. Positions 53 to 1006 are CDS-B6R (encoding amino acids 1-317 of the monkeypox virus B6R protein), positions 1007 to 1132 are 3'-UTR, and positions 1133 to 1252 are Poly A tails.

[0114] mRNA-M1038 sequence (SEQ ID No. 15):

[0115] From the 5' end, positions 1 to 52 are 5'-UTR, and positions 49-52 are Kozak sequences. Those skilled in the art can select and replace Kozak sequences that are different from those of this invention using conventional techniques. For example, the Kozak sequence can be replaced with GCC, ACC, GCCACC, or GCCANN. Positions 53 to 1072 are CDS-B6R (positions 53-121 encode the tPA signal peptide, and positions 122-1072 encode amino acids 1-317 of the monkeypox virus B6R protein). Positions 1073 to 1198 are 3'-UTR, and positions 1199 to 1318 are Poly A tails.

[0116] II. In vitro synthesis of monkeypox virus mRNA vaccine

[0117] 1. The designed monkeypox virus mRNA vaccine sequence was used to synthesize a DNA template and cloned into the PVAX1 vector to obtain the template DNA plasmid for the mRNA.

[0118] 2. The template DNA plasmid was transformed into competent DH5α cells, and a large number of amplified bacterial cells were obtained by culturing the host *E. coli*. The recombinant plasmid amplified from the amplified bacterial cells was extracted using an endotoxin-free plasmid extraction kit (Tiangen Biotech (Beijing) Co., Ltd., DP117).

[0119] The amplified recombinant plasmid was linearized: the extracted recombinant plasmid was linearized by Bsa I enzyme digestion, purified to obtain a template suitable for in vitro mRNA synthesis, and then processed using Qubit. TM Quantification was performed using the dsDNA BR Assay Kit (Invitrogen, Q32850).

[0120] 3. Using the linearized DNA prepared in step 2 as a template for in vitro mRNA synthesis, the reaction system shown in Table 1 was prepared, and in vitro transcription was performed at 37°C for 1 h to obtain a large amount of in vitro transcribed RNA. This RNA was then processed using Qubit. TM Quantification was performed using the dsDNA BR Assay Kit (Invitrogen, Q32850).

[0121] Table 1. In vitro transcription reactions were performed using the T7-FlashScribe™ Transcription kit (Cellscript, C-ASF3507).

[0122]

[0123] 4. Purify the in vitro transcribed RNA product from step 3: Add 1 μL of RNase-FreeDNase I to the transcription reaction system and incubate at 37°C for 15 min to remove the DNA template from the in vitro transcription product system, obtaining the transcription product. Then purify the obtained transcription product using the following method:

[0124] (1) Add RNase-Free H2O to the transcription product to make up a volume of 200 μL;

[0125] (2) Add 200 μL of mixture A (water-saturated phenol: chloroform: isoamyl alcohol, v:v:v, 25:24:1), vortex for 10 s, centrifuge at 4℃ for 5 min at 13800×g, and then transfer the upper aqueous phase in the tube to a new tube.

[0126] (3) Add an equal volume of mixed solution B (chloroform:isoamyl alcohol, v:v, 24:1) to the new tube, vortex for 10s, centrifuge at 4℃ for 5min at 13800×g, and then transfer the upper aqueous phase in the tube to the new tube.

[0127] (4) Add an equal volume of 5M ammonium acetate solution to the new tube, vortex to mix, place on ice for 15 min, centrifuge at 4℃ and 13800×g for 15 min, and discard the supernatant.

[0128] (5) After cleaning the RNA with 70% ice-cold ethanol, discard the 70% ethanol; resuspend the RNA in an appropriate amount of RNase-free water (Solarbio, R1600) and use a Qubit algorithm. TM Quantification was performed using the RNA BR Assay Kit (Invitrogen, Q10211).

[0129] 5. Perform an mRNA capping reaction on the RNA transcription purification product obtained in step 4. The specific steps are as follows:

[0130] (1) RNA denaturation: Take 60 μg of the purified transcription product, incubate at 65℃ for 15 min for denaturation treatment, and then transfer to ice.

[0131] (2) mRNA capping reaction: After adding the RNA denaturation product, the reaction system was prepared according to Table 2 and incubated at 37℃ for 0.5h to obtain the capped product with the Cap 1 structure of mRNA.

[0132] Table 2. Preparation of the Cap 1 capping reaction system for mRNA using ScriptCap. TM Cap 1 Capping System Kit (Cellscript, C-SCCS1710)

[0133]

[0134]

[0135] 6. Purification of mRNA capping product: Same as step 4.

[0136] 7. mRNA quality analysis:

[0137] The quality analysis of synthesized mRNA was performed using an Agilent 2100 Bioanalyzer and an RNA Nano 6000 Assay Kit (Agilent, 5067-1511). The specific steps are as follows:

[0138] (1) mRNA denaturation: denature the mRNA and mRNA ladder at 70°C for 2 min, and then immediately place them on ice.

[0139] (2) Prepare the gel: Add the RNA gel matrix to the filter tube according to the instructions, centrifuge at 1500×g at room temperature for 10 min, and store at 4℃ for later use.

[0140] (3) Prepare the gel-dye mixture: equilibrate the RNA dye in the dark for 30 min, then vortex for a few seconds, and after instantaneous centrifugation, prepare the gel-dye mixture at a ratio of 65:1. Vortex the mixture to mix well, and centrifuge at 13000×g at room temperature for 10 min.

[0141] (4) Loading the gel-dye mixture: Before using the RNA nano chip, adjust the chip preparation clamp to the top position. Place the RNA chip into the chip slot and add 9 μL of gel-dye mixture (avoiding air bubbles) to the well marked with black G; close the syringe plunger when the plunger is at the 1 mL position, press the syringe, hold it with the clamp for 30 seconds, then release it, and pull the plunger back to the 1 mL position after 5 seconds; open the syringe plunger and add 9 μL of gel-dye mixture to the other well marked with white G.

[0142] (5) Add Marker: Add 6 μL of RNA Marker to each of the sample wells and the ladder wells.

[0143] (6) Loading Ladder and mRNA: Add 1 μL of ladder to the well marked with a ladder pattern, add mRNA to the remaining 12 wells (unused wells can be replaced with RNase-free water), place the chip on a chip vortex oscillator, oscillate at 2400 r / min for 1 min, and then place the chip in an Agilent 2100 instrument for detection within 5 min.

[0144] The results are as follows Figure 2 As shown, the results indicate that the in vitro synthesized monkeypox mRNA-M351 ( Figure 2 (as shown in A29L-A and A29L-B), mRNA-M564 ( Figure 2 As shown in A35R-A), mRNA-M630 ( Figure 2 As shown in A35R-B), mRNA-M837 ( Figure 2 M1R-A and M1R-B (as shown in the image), mRNA-M972 ( Figure 2 (as shown in B6R-A) and mRNA-M1038 ( Figure 2 The bands shown in B6R-B are consistent with the target bands, with concentrations of 2083 ng / μL, 1994 ng / μL, 2220 ng / μL, 1920 ng / μL, 1982 ng / μL and 1946 ng / μL, respectively.

[0145] III. Cellular Transfection of mRNA and Detection of Protein Expression

[0146] 1. Cell seeding: 293T cells (ATCC) were seeded into 12-well plates at a density of 3 × 10⁶ cells per well. 5 Transfection can be performed when cells reach 80-90% confluency in a 37°C, 5% CO2 incubator.

[0147] 2. Preparation of the transfection complex: using Lipofectamine TM 2000 Transfection Reagent (Invitrogen, 11668-027): 49 μL Opti-MEM + 1 μL Lipo2000, 49 μL Opti-MEM + 1 μg mRNA. After standing for 5 min, the two tubes were mixed and incubated at room temperature for 20 min to form a transfection complex.

[0148] 3. Transfecting cells: Add the transfection complex to the cells and shake them up and down and left and right to distribute the transfection complex evenly. Incubate at 37°C and 5% CO2 for 18 hours and then collect the cells. There is no need to change the cell culture medium before and after transfection.

[0149] 4. Extraction of total cellular protein: After washing cells twice with PBS, lyse them thoroughly using RIPA (Jinpule, PO6M11) + 100× protease inhibitor (Jinpule, PO1C01) in cell lysis buffer by vortexing. After incubating on ice for 30 min, centrifuge at 13800×g for 15 min at 4°C and collect the supernatant.

[0150] 5. Total cellular protein quantification: Total protein in the cell lysis supernatant was quantified using the BCA protein quantification kit (Jinpulai, P06M16). The cell lysis supernatant was mixed with BCA working solution, incubated at 37°C for 45 min, and the absorbance was measured at A562 nm to calculate the total cellular protein concentration.

[0151] 6. Western blotting (WB) detection of target protein expression: using a precast protein electrophoresis gel (Bolt) TM Total protein (10 μg) was separated by 4 to 12% Bis-Tris, 1.0 mm, Mini Protein Gel (Invitrogen, NW04120BOX) electrophoresis (200 V, 22 min). The separated proteins on the gel were transferred to iBlot 2 Transfer Stacks, PVDF (Invitrogen, IB24001) membranes under gradient voltage (20 V, 1 min; 23 V, 4 min; 25 V, 2 min), and then incubated at room temperature in 1×TBST containing 5% skim milk powder at 20 rpm for 1 h.

[0152] The primary antibody was diluted 1:2000 with mouse anti-His-tag mAb (Abclone, AE003) and incubated at 20 rpm for 2 h at room temperature. The membrane was washed with 1×TBST and incubated at 60 rpm for 10 min at room temperature, repeated three times to completely remove any primary antibody residue. The secondary antibody was horseradish peroxidase (HRP)-labeled goat anti-mouse IgG diluted 1:2000 with HRPPoat anti-Mouse IgG (H+L) (Abclone, AS003) and incubated at 20 rpm for 1 h at room temperature. The membrane was washed with 1×TBST and incubated at 60 rpm for 10 min at room temperature, repeated three times to completely remove any secondary antibody residue.

[0153] HRP-labeled antibody-bound antigen was detected using an ECL chemiluminescence ultrasensitive colorimetric kit (Yisheng Biotechnology, 36208ES60) and incubated with the membrane at room temperature in the dark for 3 min. The membrane was then exposed to light using a chemiluminescence analyzer. Bands and protein markers were visualized by exposure using PageRuler. TM Prestained Protein Ladder (Invitrogen, 26617) band alignment was used to verify the expression of the target antigen protein. Antibodies on the membrane were removed using membrane regeneration solution (Solepro, SW3020), and the membrane was blocked again before incubation with an internal control antibody to check the consistency of protein loading. The primary antibody was β-actin rabbit monoclonal antibody dilution (1:50000), ACTBRabbit mAb (Abclonal, AC038), and the secondary antibody was horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody dilution (1:10000), Goat Anti-Rabbit IgG Secondary Antibody (HRP) (Sinosure, SSA004).

[0154] The results are as follows Figure 3 As shown, the mRNA-A-mix group includes mRNA-M351, mRNA-M564, mRNA-M837, and mRNA-M972; the mRNA-B-mix group includes mRNA-M351, mRNA-M630, mRNA-M837, and mRNA-M1038. Results showed that after transfection of HEK293T cells with the in vitro synthesized monkeypox virus mRNAs-M351, mRNA-M564, mRNA-M630, mRNA-M837, mRNA-M972, and mRNA-M1038, Western blotting detected highly expressed monkeypox virus A29L, A35R, M1R, and B6R target antigens, with expression levels superior to the mRNAs before sequence optimization.

[0155] IV. Preparation of Multivalent Monkeypox mRNA Vaccine

[0156] (1) Preparation of LNP-mRNA lipid nanoparticle vaccines

[0157] Ionizable cationic lipid materials (such as SM102), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and DMG-PEG2000 ethanol were completely dissolved. The lipid material ethanol solution was mixed at a molar ratio of 50:10:38.5:1.5 and then mixed with 20 mM sodium citrate buffer (pH 4.0) of group A mRNA (mRNA-M351, mRNA-M564, mRNA-M837, and mNRA-M972) and group B mRNA (mRNA-M351, mRNA-M630, mRNA-M837, and mRNA-M1038) at a volume ratio of 1:3 (lipid material:mRNA) at a flow rate of 12 mL / min in the Myanna Nanomedicine Preparation System. The collected sample solution was diluted 10-fold in DPBS buffer and then concentrated by ultrafiltration through a 50 kDa PES tube at 4°C and 2000 × g to remove ethanol. Finally, the vaccine formulation that had passed through a 0.22 μm filter was adjusted to the appropriate concentration with DPBS buffer for further experiments.

[0158] (2) Preparation of YK009 LNP-mRNAs lipid nanoparticle vaccine

[0159] The novel cationic ester compound (YK009), 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and DMG-PEG2000 were completely dissolved in ethanol. The novel cationic ester compound 2-octyldecyl 6-((4-(decoxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (YK009), CH40H79NO5 (structural formula shown below (I)), was obtained by dissolving decyl 4-((2-hydroxyethyl)amino)butyrate and 2-octyldecyl 6-bromohexanoate in acetonitrile, adding potassium carbonate and potassium iodide, heating to 70°C and stirring for 20 h, cooling to room temperature, filtering, concentrating the filtrate under vacuum to remove the solvent, and purifying the residue by silica gel chromatography to obtain the cationic ester compound. The above-mentioned lipid material ethanol solution was mixed at a molar ratio of 50:10:38.5:1.5 and then mixed with 20mM sodium citrate buffer (pH 4.0) of group A mRNA (mRNA-M351, mRNA-M564, mRNA-M837, and mNRA-M972) and group B mRNA (mRNA-M351, mRNA-M630, mRNA-M837, and mRNA-M1038) at a volume ratio of 1:3 (lipid material:mRNA) at a flow rate of 12 mL / min in the Myanna Nanomedicine Preparation System. The collected sample solution was diluted 10-fold in DPBS buffer and then concentrated by ultrafiltration through a 50 kDa PES tube at 4°C and 2000 × g to remove ethanol. Finally, the vaccine formulation that had passed through a 0.22 μm filter membrane was adjusted to the appropriate concentration with DPBS buffer for further experiments.

[0160]

[0161] Example 2: Serum antibody detection in mice immunized with monkeypox virus mRNA vaccine

[0162] I. Muscle immunization in mice with a multivalent monkeypox virus mRNA vaccine:

[0163] BALB / c mice (female, 6-8 weeks old, 16-18g, Vital River Pharmaceuticals, Beijing) were randomly divided into four groups: monkeypox virus LNP-mRNAs-A group (mRNA = 40 μg / mouse), LNP-mRNAs-B group (mRNA = 40 μg / mouse), YK009-mRNAs-A group (mRNA = 40 μg / mouse), YK009-mRNAs-B group (mRNA = 40 μg / mouse), and a negative control group (PBS, pH = 7.4) (n=5 per group, fed normally). Mice were immunized intramuscularly. Immunization was performed twice, and sufficient mouse serum was obtained by orbital blood collection on day 10 post-immunization.

[0164] II. Muscle immunization and serum antibody detection in mice using a multivalent monkeypox virus mRNA vaccine

[0165] BALB / c mice (female, 6-8 weeks old, 16-18g, Vital River Pharmaceuticals, Beijing) were randomly divided into different immunization regimens: LNP-mRNAs-A (Group A: mRNA-M351, mRNA-M564, mRNA-M837, and mRNA-M972; mRNA = 40 μg / mouse), LNP-mRNAs-B (Group B: mRNA-M351, mRNA-M630, mRNA-M837, and mRNA-M1038; mRNA = 40 μg / mouse), and Y... Mice were immunized via intramuscular injection in immunization groups (5 mice per group, fed normally) of K009-mRNAs-A (group A: mRNA-M351, mRNA-M564, mRNA-M837, and mRNA-M972; mRNA = 40 μg / mouse) and YK009-mRNAs-B (group B: mRNA-M351, mRNA-M630, mRNA-M837, and mRNA-M1038; mRNA = 40 μg / mouse) and a negative control group (PBS, pH = 7.4). Two immunizations were administered, with a 14-day interval between the LNP-mRNA group and a 21-day interval between the YK009-mRNA group. Sufficient mouse serum was obtained by orbital blood collection on day 10 after each immunization. The production of monkeypox virus antigen-specific binding antibody IgG in the mouse immune serum was detected by ELISA to evaluate the in vivo immunogenicity of the monkeypox virus vaccine.

[0166] The production of monkeypox virus antigen-specific binding antibody IgG in the serum of immunized mice was detected by ELISA. The specific detection method is as follows:

[0167] (1) Coating: A29L, A35R, B6R, and M1R proteins were diluted to 1 ng / μl with carbonate buffer (50 mM, pH 9.6, filtered through a 0.22 μm filter). 100 ng of RBD protein dilution was added to each well of a 96-well plate, sealed, and incubated overnight at 4°C.

[0168] (2) Washing: After coating overnight, pour out the 96-well plate to remove the protein coating solution, add 200 μL of washing buffer (containing 0.2% Tween-20 and 1×TBS) to each well, gently shake by hand for 30 seconds and then pat dry on paper. Repeat 6 times.

[0169] (3) Blocking: Add 200 μl of blocking solution (1×TBS containing 2% BSA) to each well and incubate at 37°C for 2 h;

[0170] (4) Washing the plate: Pour out the 96-well plate to remove the blocking solution, add 200 μl of washing solution (containing 0.2% Tween-20 and 1×TBS) to each well, gently shake by hand for 30 seconds, and then pat dry on paper. Repeat 6 times.

[0171] (5) Incubation with primary antibody: The serum of immunized mice was serially diluted 10-fold with antibody diluent (washing solution containing 0.5% BSA) to obtain serum at 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.

[0172] (6) Washing the plate: Pour out the 96-well plate to remove the serum diluent, add 200 μL of washing buffer (containing 0.2% Tween-20 and 1×TBS) to each well, gently shake by hand for 30 seconds, and then pat dry on paper. Repeat 6 times.

[0173] (7) Incubation of secondary antibody: horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (Beyotime, A0216) was diluted 250 times with antibody dilution buffer (washing buffer containing 0.5% BSA) to obtain secondary antibody dilution buffer. 100 μl of secondary antibody dilution buffer was added to each well and incubated at 37°C for 1 h.

[0174] (8) Washing the plate: Pour out the 96-well plate to remove the secondary antibody dilution solution, add 200 μl of washing buffer (containing 0.2% Tween-20 and 1×TBS) to each well, gently shake by hand for 30 seconds, and then pat dry on paper. Repeat 6 times.

[0175] (9) Color development: Add 100 μl of TMB substrate (Tiangen, RA107) to each well and incubate at room temperature in the dark for 20 min;

[0176] (10) Terminate color development: Add 50 μL of 2M H2SO4 to each well and detect the OD value of A450 on a microplate reader;

[0177] (11) Determination of serum binding antibody IgG titer: If the OD / negative control OD of a certain dilution is ≥2.1, the OD / negative control OD of the next dilution is <2.1. This dilution factor is the antibody titer corresponding to the serum sample (if the negative control OD is <0.05, it is calculated as 0.05).

[0178] The results are as follows Figure 4 The results showed that high titers of antigen-specific binding antibodies IgG were detected in the serum of mice immunized with monkeypox virus mRNA vaccines LNP-mRNAs-A and LNP-mRNAs-B. The average binding antibody IgG titer of A29L was approximately 1:48000 (group A) and 1:92000 (group B) (results are shown in the figure). Figure 4 (As shown in Figure 1); the average M1R binding antibody IgG titer was approximately 1:4040000 (Group A) and 1:3600000 (Group B) ( Figure 4 (As shown in Figure 2); the average titer of B6R-binding antibody IgG was approximately 1:900,000 (Group A) and 1:600,000 (Group B). Figure 4 (As shown in Figure 3); the average antibody-IgG titer of A35R binding was approximately 1:1080000 (Group A) and 1:1200000 (Group B). Figure 4 (As shown in Figure 4); the average binding antibodies in the serum of mice immunized with YK009-mRNAs-A and YK009-mRNAs-B were: A29L: 1:64000 (Group A), 1:10000 (Group B); A35R: 1:1000000 (Group A), 1:820000 (Group B); M1R: 1:1000000 (Group A), 1:100000 (Group B); B6R: 1:1000000 (Group A), 1:1000000 (Group B) Figure 4 (As shown in Figure 5).

[0179] III. Detection of neutralizing antibodies against live vaccinia Tiantan strain vaccinia virus in the serum of mice immunized with monkeypox virus mRNA vaccine.

[0180] The neutralizing effect of mouse immune serum on live poxvirus and the determination of neutralizing antibody titer (NT) were evaluated using live vaccinia Tiantan strain vaccinia virus. 50 This allows for the evaluation of the in vivo immunogenicity of monkeypox virus LNP-mRNAs-A and LNP-mRNAs-B vaccines.

[0181] vaccinia virus neutralizing antibody NT 50 Potency determination:

[0182] The neutralizing effect of live vaccinia Tiantan strain vaccinia virus on the serum of mice immunized with monkeypox virus mRNA vaccine was evaluated. The live vaccinia Tiantan strain vaccinia virus used in the evaluation was provided by the National Institutes for Food and Drug Control (NIFDC). Specific detection methods are as follows:

[0183] Mouse immune serum was serially diluted 3-fold from 1 / 30 using DMEM complete medium to obtain 6 different serum dilutions, which were then mixed with 650×TCID60. 50 The pseudovirus was incubated at 37°C. A pseudovirus control group without pseudovirus and a pseudovirus control group without serum samples were also included. After 1 hour of incubation, 2 × 10⁻⁶ cells were added to each well. 4 Vero cells were cultured at 37°C and 5% CO2. Since the Tian Tan strain of live virus expresses firefly luciferase upon entering the cells, it reacted with a luminescent substrate after 48 hours, and luminescence was detected. The percentage of virus inhibition was calculated by comparing the luminescence value with that of the virus control group. The half-inhibitory dilution (WIC) was calculated by using a formula to determine the serum dilution factor required for 50% inhibition of the pseudovirus. The WIC is expressed as the half-neutralizing dilution (NT). 50This refers to the neutralizing activity of serum antibodies against the virus.

[0184] The results are as follows Figure 5 The results showed that the serum of mice immunized with monkeypox virus LNP-mRNAs-A group / LNP-mRNAs-B group vaccines had a neutralizing effect on live vaccinia Tiantan strain vaccinia virus. After two immunizations, the average virus-neutralizing antibody titers (NT50) were ~1:3276 and ~1:2066, respectively.

[0185] The inventors discovered that the mRNA vaccine prepared using unoptimized mRNA as raw material, according to the preparation method of the multivalent monkeypox mRNA vaccine in Example 1, had almost undetectable antigen-specific IgG and neutralizing antibody titers.

[0186] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions 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 selected from the mRNA encoding monkeypox virus A29L, the mRNA encoding monkeypox virus A35R, the mRNA encoding monkeypox virus M1R, or the mRNA encoding monkeypox virus B6R. The mRNA sequence encoding monkeypox virus A29L is shown in SEQ ID NO.10; The mRNA sequence encoding monkeypox virus A35R is shown in SEQ ID NO.11 or SEQ ID NO.12; The mRNA sequence encoding monkeypox virus M1R is shown in SEQ ID NO.13; The mRNA sequence encoding monkeypox virus B6R is shown in SEQ ID NO.14 or SEQ ID NO.

15.

2. An mRNA combination, characterized in that, include: mRNA encoding monkeypoxvirus A29L, mRNA encoding monkeypoxvirus A35R, mRNA encoding monkeypoxvirus M1R and mRNA encoding monkeypoxvirus B6R; The mRNA sequence encoding monkeypox virus A29L is shown in SEQ ID NO.10; The mRNA sequence encoding monkeypox virus A35R is shown in SEQ ID NO.11 or SEQ ID NO.12; The mRNA sequence encoding monkeypox virus M1R is shown in SEQ ID NO.13; The mRNA sequence encoding monkeypox virus B6R is shown in SEQ ID NO.14 or SEQ ID NO.

15.

3. A biomaterial, characterized in that, Includes any one of the following: B1) to B6): B1) A nucleic acid molecule encoding the mRNA of claim 1, or a nucleic acid molecule encoding a combination of the mRNAs of claim 2; 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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); 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).

4. The use of the mRNA of claim 1 or the combination of mRNAs of claim 2 in the preparation of monkeypox vaccine.

5. An mRNA complex, characterized in that, It includes a delivery vector and the mRNA of claim 1 or the combination of the mRNA of claim 2.

6. The mRNA complex according to claim 5, characterized in that, The delivery carrier includes any one of ionizable liposomes, cationic liposomes, ionizable proteins, cationic proteins, ionizable polymers, cationic polymers, ionizable micelles, or cationic micelles.

7. The mRNA complex according to claim 5, characterized in that, The delivery carrier includes YK009, 1,2-distearate-sn-glycerol-3-phosphocholine, cholesterol, and DMG-PEG2000.

8. A monkeypox mRNA vaccine, characterized in that, Includes the mRNA of claim 1, the mRNA combination of claim 2, or the mRNA complex of any one of claims 5-7; In the mRNA combination, when the sequence of the mRNA encoding monkeypox virus A35R is as shown in SEQ ID NO.11, the sequence of the mRNA encoding monkeypox virus B6R is as shown in SEQ ID NO.

14. Alternatively, when the sequence of the mRNA encoding monkeypox virus A35R is as shown in SEQ ID NO.12, the sequence of the mRNA encoding monkeypox virus B6R is as shown in SEQ ID NO.

15.

9. A pharmaceutical composition, characterized in that, It includes at least one of the mRNA of claim 1, the mRNA combination of claim 2, the biomaterial of claim 3, the mRNA complex of any one of claims 5-7, or the monkeypox mRNA vaccine of claim 8, and optionally a pharmaceutical carrier.

10. A reagent kit, characterized in that, It includes at least one of the mRNA of claim 1, the mRNA combination of claim 2, the biological material of claim 3, the mRNA complex of any one of claims 5-7, or the monkeypox mRNA vaccine of claim 8.

Images