CN119570817B

CN119570817B - Multi-antigen monkey pox RNA vaccine and preparation method thereof

Info

Publication number: CN119570817B
Application number: CN202510142236.7A
Authority: CN
Inventors: 王升启; 杨静; 左军; 张震; 龙晋蓉
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2025-02-10
Filing date: 2025-02-10
Publication date: 2025-06-06
Anticipated expiration: 2045-02-10
Also published as: CN119570817A

Abstract

本发明涉及多抗原猴痘RNA疫苗及其制备方法，涉及核酸疫苗技术领域。所述多抗原mRNA包含编码猴痘病毒的抗原性多肽或其抗原性片段、变体或衍生物的序列，所述抗原性多肽或其抗原性片段至少包含猴痘病毒的细胞内成熟病毒和细胞外囊膜病毒的病毒蛋白。本发明提供了一种针对猴痘病毒的多抗原mRNA疫苗及序列，本发明的mRNA在体外细胞中能够表达抗原蛋白，且免疫小鼠后能够检测到高效价的抗猴痘病毒A29L、A33L、B6R、M1R和E8L蛋白IgG，并产生高效价的抗牛痘天坛株活病毒中和抗体，具有良好的免疫原性，对于猴痘病毒的预防有重要意义。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

Multi-antigen monkey pox RNA vaccine and preparation method thereof

Technical Field

The invention relates to the technical field of nucleic acid vaccines, in particular to a multi-antigen monkey pox virus mRNA vaccine containing multiple mRNAs and a preparation method thereof.

Background

The popularization of safe and effective vaccines worldwide is the strongest weapon against monkey poxviruses and is also the key to the termination of the monkey poxepidemic. So far, the monkey pox has no specific vaccine temporarily, the monkey pox virus and the smallpox virus and the vaccinia virus belong to the same genus of orthopoxvirus, the smallpox vaccine can provide cross protection, and the effective rate of the vaccinated smallpox vaccine for preventing the monkey pox is 85 percent or the severity degree after infection is reduced. There are only two currently approved by the FDA for use in vaccines against monkey poxviruses, ACAM2000 and JYNNEOS. However, these vaccines cause rare side effects such as myocarditis and pericarditis, with high impact on eczema patients and pregnant women. Thus, there remains a need to develop an effective and safe new generation of monkey pox specific vaccines. And the public health crisis continuously caused by the monkey poxvirus is still not relieved yet under the condition that the monkey poxvirus is abused.

The monkey poxvirus is an enveloped double-stranded DNA virus that shares two distinct genetically evolved branches—the mid-non-branched and the western non-branched. Of these, western non-branching mortality is about 3.6%, and the disease caused by the history of the middle non-branching is more serious, and mortality is about 10.6%, and is considered to be more infectious.

The mRNA vaccine has the technical advantages of short research and development period, high productivity speed increase and the like, and is more suitable for emergency research and development of prophylactic vaccines of monkey pox viruses. The research and development of the independently controllable multi-antigen mRNA vaccine capable of preventing the infection of the monkey pox has important significance for the prevention and control of the epidemic situation of the monkey pox.

Disclosure of Invention

The invention aims to enrich the types of monkey pox virus vaccines in the prior art and provides a multi-antigen mRNA vaccine, a pharmaceutical composition and a kit aiming at the monkey pox virus. The difficulty in designing mRNA vaccines for monkey poxviruses is high, and in particular the realization of complete and efficient expression of the virus can be affected by a number of factors. The mRNA provided by the invention can express antigen protein in vitro cells, and can detect high-titer neutralizing antibodies against live viruses of vaccinia Tiantan strains after mice are immunized, so that the mRNA has good immunogenicity and is of great significance for monkey pox virus prevention.

The technical scheme provided by the invention is as follows:

In a first aspect, the invention provides an mRNA comprising an antigenic polypeptide encoding a multi-antigen monkey poxvirus or an antigenic fragment, variant or derivative thereof comprising at least viral proteins of a monkey poxvirus intracellular mature virus (Intracellular mature virion, IMV) and an extracellular enveloped virus (Extracellular enveloped virion, EEV).

In the present invention, the infectious disease monkey pox (Monkeypox, MPX) is a viral zoo disease caused by infection with a monkey pox virus (Monkeypox virus, MPXV), and cross species transmission can be transmitted to humans by animals and can be transmitted interpersonal. IMVs and EEVs express different viral proteins, forming unique antigenic features to distinguish between the two forms of virions.

In one embodiment, the antigenic polypeptide or antigenic fragment thereof comprises mature virion surface proteins a29L, M1R, E L and envelope virion surface proteins a35R and B6R that have protective immunogenicity. In the invention, the A29L protein is a monoclonal antibody target of MPXV, and mediates recognition of host cells to enable the virus to be fused with the host cells in a catalytic manner. A35R is an important component of the envelope virus particle and is also a potential detection target of serology. The presence of B6R protein on the membrane of EEV particles negatively regulates complement activation and its intracellular localization is determined by interaction with a33 or a 34. E8L has a ganglioside binding motif consisting of subunits, and the ganglioside binding domain of viral proteins is a characteristic target for antiviral drugs and neutralizing antibodies. The use of a combination of intracellular mature virions and extracellular enveloped virions provides a better protective effect.

In the present invention, the multi-antigen mRNA comprises at least both mRNA sequences encoding A29L, M1R, E8L, A R and B6R of the monkey poxvirus.

In one embodiment, the mRNA has a nucleotide sequence as shown in one of the sequences shown in SEQ ID No.1 to SEQ ID No.6, or a sequence which has more than 90% homology with one of the sequences shown in SEQ ID No.1 to SEQ ID No.6 and is functionally identical.

In the invention, CDS of the monkey pox virus mRNA is composed of optimized codons, so that the protein can be efficiently expressed at the cellular level (the protein with higher level is expressed after the cells are transfected), and the proteins in the sequences shown in SEQ ID No.1 to SEQ ID No.6 can be efficiently expressed. After the mRNA of the invention is prepared into a vaccine (vaccine for expressing monkey pox antigen protein), the effectiveness is good and the immunogenicity is good through the verification of mouse immunity.

In a preferred embodiment, the nucleotide sequence of the mRNA has more than 95% homology and functionally identical sequence to one of the sequences shown in SEQ ID No.1 to SEQ ID No. 6.

In a more preferred embodiment, the nucleotide sequence of the mRNA has more than 99% homology and functionally identical sequence to one of the sequences shown in SEQ ID No.1 to SEQ ID No. 6.

"Homology" as used herein is synonymous with "identity" and means that the sequence used has (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 in terms of amino acid sequence or nucleotide sequence to the sequences obtained in the prior art, and still has the same function as the original amino acid sequence or the nuclear sweet potato sequence.

In one embodiment, the nucleotide sequence of the mRNA encoding both the A29L, M1R, E8L, A R and B6R domains in the monkey poxvirus protective immunogenic protein is as shown in SEQ ID No.1 to SEQ ID No. 6.

The mRNA comprises the following elements in sequence from 5' to 3' of a 5' cap structure, a 5' UTR sequence, an antigenic polypeptide of a monkey poxvirus or an antigenic fragment, variant or derivative thereof, a 3' UTR sequence and a polyadenylation tail sequence.

The mRNA-2949 DNA is shown as SEQ ID No.1, wherein the sequence from the 5' end is 5' -UTR from 1 to 52, the sequence from 49 to 52 is Kozak, the sequence from 53 to 121 is secretory peptide, the sequence from 122 to 493 is CDS-A35R (amino acid 1-124 of encoding monkey pox virus A35R protein), the sequence from 494 to 508 is flexible linker, the sequence from 509 to 1048 is CDS-M1R (amino acid 1-180 of encoding monkey pox virus M1R protein), the sequence from 1049 to 1066 is flexible linker, the sequence from 1067 to 1900 is CDS-B6R (amino acid 1-278 of encoding monkey pox virus B6R), the sequence from 1901 to 1918 is flexible linker, the sequence from 1919 to 2245 is CDS-A29L (amino acid 1-109 of encoding monkey pox virus A29L), the sequence from 2246 to 2266 is flexible linker, the sequence from 2267 to 2998 is CDS-B8L (amino acid 1-244 of encoding monkey pox virus E8L), the sequence from 1067 to 1900 is CDS-B6R (amino acid 1-278 of encoding monkey pox virus B6R), and the sequence from 3007 ' -37 is Poly-37A.

MRNA-3000 DNA sequence is shown as SEQ ID No.2, 5' -UTR from 5' end 1 to 52, kozak sequence 49-52, secretory peptide 53 to 121, CDS-A35R (amino acid 1-124 of monkey pox virus A35R protein), flexible linker 494 to 508, CDS-M1R (amino acid 1-180 of monkey pox virus M1R protein), short peptide 1049 to 1114, CDS-B6R (amino acid 1-279 of monkey pox virus B6R protein), flexible linker 1952 to 1969, CDS-A29L (amino acid 1-109 of monkey pox virus A29L protein), flexible linker 97 to 2318 to 3049, CDS-E8L (amino acid 1-244 of monkey pox virus E8L protein), CDS-B6R protein 1-279, and Poly3178 ' -3053-3198, and stop codon 3053-3178.

MRNA-2949A DNA template sequence (SEQ ID No. 3):

From the 5' end, 1 to 52 are 5' -UTR,49-52 are Kozak sequences, 53 to 121 are secretory peptides, 122 to 661 are CDS-M1R (amino acids 1-180 encoding the M1R protein of the monkey pox virus), 662 to 676 are flexible linkers, 677 to 1048 are CDS-A35R (amino acids 1-124 encoding the A35R protein of the monkey pox virus), 1049 to 1066 are flexible linkers, 1067 to 1900 are CDS-B6R (amino acids 1-279 encoding the B6R protein of the monkey pox virus), 1901 to 1918 are flexible linkers, 1919 to 2245 are CDS-A29L (amino acids 1-109 encoding the A29L protein of the monkey pox virus), 2246 to 2266 are flexible linkers, 2267 to 2998 are CDS-E8L (amino acids 1-244 encoding the E8L protein of the monkey pox virus), 2999 to 3001 are termination codons, and 3' -UTR 3 to 3247 are Poly tails.

MRNA-3000A DNA template sequence (SEQ ID No. 4):

From the 5 'end, 1 to 52 are 5' -UTR,49-52 are Kozak sequences, 53 to 121 are secretory peptides, 122 to 661 are CDS-M1R (amino acids 1-180 of the monkey poxvirus M1R protein), 662 to 676 are flexible linkers, 667 to 1048 are CDS-A35R (amino acids 1-124 of the monkey poxvirus A35R protein), 1049 to 1114 are 2A short peptides, 1115 to 1951 are CDS-B6R (amino acids 1-279 of the monkey poxvirus B6R protein), 1952 to 1969 are flexible linkers, 1970 to 2296 are CDS-A29L (amino acids 1-109 of the monkey poxvirus A29L protein), 2297 to 2317 are flexible linkers, 2318 to 3049 are CDS-E8L (amino acids 1-244 of the monkey poxvirus E8L protein), 0 to 3052 are stop codons, 313 to 3178 are PolyUTR 3 to 3298.

MRNA-2949B DNA template sequence (SEQ ID No. 5):

From the 5' end 1 to 52 are 5' -UTR,49-52 are Kozak sequences, 53 to 121 are secretory peptides, 122 to 661 are CDS-M1R (amino acids 1-180 of the monkey poxvirus M1R protein), 662 to 676 are flexible linkers, 667 to 1003 are CDS-A29L (amino acids 1-109 of the monkey poxvirus A29L protein), 1004 to 1021 are flexible linkers, 1022 to 1393 are CDS-A35R (amino acids 1-124 of the monkey poxvirus A35R protein), 1394 to 1411 are flexible linkers, 1412 to 2245 are CDS-B6R (amino acids 1-279 of the monkey poxvirus B6R protein), 2246 to 2266 are flexible linkers, 2267 to 2998 are CDS-E8L (amino acids 1-244 of the monkey poxvirus E8L protein), 2999 to 3001 are stop codons, 3002 to 3127 are 3' -UTR 8 to 3247.

MRNA-22949C DNA template sequence (SEQ ID No. 6):

from the 5' end, 1 to 52 are 5' -UTR,49-52 are Kozak sequences, 53 to 121 are secretory peptides, 122 to 661 are CDS-M1R (amino acids 1-180 of the monkey poxvirus M1R protein), 662 to 676 are flexible linkers, 667 to 1003 are CDS-A29L (amino acids 1-109 of the monkey poxvirus A29L protein), 1004 to 1021 are flexible linkers, 1022 to 1393 are CDS-A35R (amino acids 1-124 of the monkey poxvirus A35R protein), 1394 to 1411 are flexible linkers, 1412 to 2143 are flexible linkers, CDS-E8L (amino acids 1-244 of the monkey poxvirus E8L protein), 2144 to 2164 are flexible linkers, 2165 to 2998 are CDS-B6R (amino acids 1-279 of the monkey poxvirus B6R protein), 2999 to 3001 are stop codons, 3002 to 3127 are 3' -UTR 8 to 3247.

In addition, the kozak sequence can be replaced by GCC, ACC, GCCACC or GCCANN, the stop codon can be replaced by TAA or TAG, or 2 stop codons can be added, such as TGA, TAA, TAG, the length of PolyA tail can be more than 30A, or the 31 th to 40 th A can be replaced by GCATATGACT. The linker may be replaced with CDS encoding an amino acid sequence of GSGSAS, (GGGGS) n (n=1-5), GSAGSA, AAAGGAA, or the like.

In a third aspect, the present invention provides a nucleic acid molecule or protein related biomaterial as hereinbefore described comprising any one of the following B1) to B6):

B1 A nucleic acid molecule encoding said protein, said nucleic acid molecule being a DNA molecule encoding a plurality of mRNAs as described above;

B2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

B4 A recombinant plasmid containing the nucleic acid molecule of B1) or a recombinant plasmid containing the expression cassette of B2);

b5 A recombinant microorganism comprising B1) said nucleic acid molecule, or a recombinant microorganism comprising B2) said expression cassette, or a recombinant microorganism comprising B3) said recombinant vector;

B6 A transgenic cell line comprising the nucleic acid molecule of B1), or a transgenic cell line comprising the expression cassette of B2), or a transgenic cell line comprising the recombinant vector of B3).

In the invention, the coding sequence of the protein can be cloned into a plasmid by a genetic engineering technology to carry out in vitro transcription for mRNA synthesis, and the method preferably comprises 1) cloning a DNA fragment corresponding to the mRNA into an 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 amplified recombinant cell, and carrying out PCR amplification to obtain a DNA template for in vitro mRNA expression, and 3) constructing an RNA in vitro synthesis system comprising the DNA template to carry out in vitro synthesis of the mRNA to obtain the mRNA of the active ingredient. In the present invention, the specific sequence of the DNA fragment can be determined according to the base complementary pairing rules.

In one embodiment, after in vitro transcription, the transcribed RNA product is subjected to a capping reaction, and the resulting mRNA is linked at its 5' end to a Cap (Cap-1) structure.

In one embodiment, the recombinant vector is vector pVAX1.

In one embodiment, the recombinant vector is transferred into a cell expressing the viral protein for expression, preferably the cell is selected from the group consisting of HEK293T cells, 293FTX cells, HEK293A cells.

In a fourth aspect, the invention also provides an mRNA-lipid complex comprising a delivery vehicle and the mRNA as described above;

Preferably, the delivery vehicle comprises any one of an ionizable liposome, a cationic liposome, an ionizable protein, a cationic protein, an ionizable polymer, a cationic polymer, an ionizable micelle, a cationic micelle, an ionizable lipid nanoparticle, a cationic lipid nanoparticle;

More preferably, the delivery vehicle is selected from the group consisting of ionizable Lipid Nanoparticles (LNPs).

In one embodiment, the mRNA-lipid complex is selected from the group consisting of an ionizable lipid-mRNA complex, a cationic lipid-mRNA complex, or a novel cationizable lipid-mRNA complex, an ionizable lipid-mRNA lipid nanoparticle, a cationic lipid-mRNA lipid nanoparticle, or a novel cationizable lipid-mRNA lipid nanoparticle;

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-phosphate ethanolammonium and/or cholesterol.

Preferably, the ionizable lipid-mRNA complex nanoparticle, cationic lipid-mRNA lipid nanoparticle, or novel cationizable lipid-mRNA lipid nanoparticle further comprises a pegylated lipid, 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine, and cholesterol.

In one embodiment, the mRNA-lipid complex comprises lipid nanoparticles LNP-mRNA prepared from mRNA binding ionizable lipids, cationizable lipid material-mRNA lipid nanoparticles prepared from mRNA binding ionizable lipids SM 102.

In one embodiment, the mRNA-Lipid complex is prepared by mixing the mRNA with an ionizable Lipid material and packaging the mixture with a Lipid, wherein the ionizable Lipid material can be YK009, MC3, SM102, ALC0315, lipid 5, DOTAP, etc.;

in a preferred embodiment, the preparation method comprises dissolving and mixing an ionizable lipid with 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, DMG-PEG2000, and then mixing mRNA with the mixed lipid.

In a fifth aspect, the present invention provides a multi-antigen monkey poxvirus mRNA vaccine comprising the mRNA described above, the biological material described above or the mRNA-lipid complex described above;

preferably, the mRNA vaccine induces the cells to produce virus-like particles, and/or the mRNA vaccine further comprises an adjuvant.

The term "adjuvant" refers to an agent that increases, stimulates, activates, boosts or modulates an immune response against an active ingredient of the composition at a cellular or humoral level.

Through a plurality of experiments, the inventor finally discovers that the specific combination of the specific framework sequences and the coding sequences can enable the prepared vaccine to achieve better immunogenicity and stability.

The multi-antigen monkey pox virus mRNA vaccine is a vaccine against monkey pox virus, which is administered to subjects including, but not limited to, mammals and humans. The mammal includes, but is not limited to, a monkey, camel, cow, horse, goat, sheep, pig, cat, dog, rabbit, mouse, or rat, etc. Preferably, the vaccine is an infectious disease vaccine for preventing infection by a monkey poxvirus. "prevention" as used herein refers to all actions of avoiding symptoms or delaying stress of a particular symptom by administering the product of the present invention before or after the onset of disease.

In a sixth aspect, the invention provides a pharmaceutical composition comprising the aforementioned mRNA, the aforementioned biological material or the aforementioned mRNA-lipid complex and/or the aforementioned multi-antigen mRNA vaccine, and optionally a pharmaceutically acceptable carrier. The invention provides the use of a product comprising the aforementioned mRNA, the aforementioned biological material or the aforementioned mRNA-lipid complex and/or the aforementioned mRNA vaccine for the preparation of a medicament for the prevention and/or treatment of a monkey pox virus infection.

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.

In the present invention, the term "neutralizing antibody" generally means that a microorganism is stimulated to produce a large number of antibodies after invading the human body, but only a part of the antibodies can rapidly recognize the microorganism and "catch" the microorganism before invading the cells of the human body, thereby protecting the human body from infection. This process is called neutralization, and the antibody that acts is a neutralizing antibody.

In the invention, the lipid nanoparticle LNP-mRNA vaccine preparation prepared from the ionizable lipid can realize the expression of the in vitro cell monkey pox virus immunogenic antigen protein, and the lipid nanoparticle LNP-mRNA vaccine preparation prepared from the cationizable lipid SM102 can realize the expression of the in vitro cell monkey pox virus immunogenic antigen protein.

The invention has the beneficial effects that:

The successful development of the mRNA vaccine is greatly dependent on the optimization of the sequence of the mRNA, the mRNA serving as the active ingredient of the vaccine provided by the invention consists of 1083 skeleton code sequences and different antigen code sequences, the mRNA sequence is mRNA subjected to codon optimization, HEK293T cells are transfected through the commercial transfection reagent package transfection, and the mRNA vaccine can simultaneously express a large amount of antigen proteins in the cells, namely has good antigen expression efficacy in vitro cells.

The multi-antigen mRNA vaccine provided by the invention can realize stable and safe expression and effective activation of immune response in vivo, can cause neutralizing antibody response, and can detect that the average NT ₅₀ of neutralizing antibodies of high-titer anti-vaccinia Tiantan strain live viruses in immunized mice serum is mRNA-2949:2323, mRNA-3000:2185, mRNA-2949 A:1:5938, mRNA-3000 A:1:5042, mRNA-2949 B:1:5053 and mRNA-2949 C:1:6163 respectively, wherein the neutralizing antibody level induced by the mRNA-2949A and the mRNA-2949C is higher than that of other groups, so that the mRNA-2949A, mRNA-2949C has good immunogenicity.

Drawings

FIG. 1 is a schematic diagram of the structures of active components mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000, A, mRNA-2949B and mRNA-2949C of the monkey pox virus mRNA vaccine provided by the invention;

FIG. 2 is a graph showing the results of mass analysis in example 1 of the present invention;

FIG. 3 is a graph showing the results of detection of the expression of a transfected protein by cells in example 1 of the present invention;

FIG. 4 shows the detection of specific antibodies IgG in serum after immunization of mice in example 2 of the present invention;

FIG. 5 is a graph showing the detection result of an immune mouse serum anti-vaccinia Tiantan strain live virus neutralizing antibody NT ₅₀ in example 2 of the present invention;

FIG. 6 is a reaction system recipe for the in vitro transcription step of example 1;

FIG. 7 shows the formulation of the reaction system for the capping reaction step of example 1.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Example 1 sequence design, preparation of monkey poxvirus mRNA vaccine and detection of antigen expression in vitro cells

1. Sequence design of monkey pox mRNA vaccine

The sequence design of the monkey pox mRNA vaccine adopts an optimized mRNA framework code sequence to strengthen the stability and the protein expression efficiency of mRNA. The CDS of the monkey poxvirus mRNA is composed of optimized codons, determining the amino acid sequence of the monkey poxvirus IMV and EEV with protective immunogenic proteins.

To achieve in vitro transcription of mRNA, a monkey poxvirus mRNA vaccine sequence template was constructed on vector PVAX1 (the sequence between the last 3 bases GGG of the T7 promoter and the XhoI site of the multiple cloning site was replaced with the template sequence of mRNA, and the BsaI cleavage site sequence was inserted between the mRNA template sequence and XhoI cleavage site to give a recombinant plasmid, the other sequences being unchanged), and in vitro transcription was initiated by T7 transcriptase using the T7 promoter sequence.

Thus, the design and optimization result in monkey pox mRNA vaccine active ingredients mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000, A, mRNA-2949B and mRNA-2949C. As shown in fig. 1. The sequences of mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000A, mRNA-2949B and mRNA-2949C are respectively shown as sequences 1,2, 3,4,5 and 6 in the sequence table.

2. In vitro synthesis of monkey poxvirus mRNA vaccine

1. And (3) synthesizing a DNA template from the designed monkey poxvirus mRNA vaccine sequence, and cloning the DNA template onto a PVAX1 vector to obtain a template DNA plasmid of mRNA.

2. The competent cells DH5 alpha are transformed by the template DNA plasmid, and a large amount of amplified thalli are obtained by culturing host escherichia coli. Recombinant plasmids amplified in the amplified cells were extracted by means of endotoxin-free plasmid large extraction kit (Tiangen Biotechnology (Beijing) Co., ltd., DP 117).

The amplified recombinant plasmid was linearized by enzyme-cutting the extracted recombinant plasmid with Bsa I enzyme, purified to obtain a template for in vitro synthesis of mRNA, and quantified using QubitTM dsDNA BR Assay Kit (Invitrogen, Q32850) kit.

3. Preparing a reaction system shown in fig. 6 by taking the linearized DNA prepared in the step 2 as an in vitro synthesis template of mRNA, incubating for 3h at 37 ℃ for in vitro transcription to obtain a large amount of in vitro transcribed RNA, and quantifying by adopting a QubitTM dsDNA BR Assay Kit (Invitrogen, Q32850) kit.

4. And (3) purifying the in vitro transcribed RNA product in the step (3), namely adding 1 mu L of RNase-FREE DNASE I into a transcription reaction system, and incubating for 15min at 37 ℃ to remove a DNA template in the in vitro transcription product system, so as to obtain a transcription product. And purifying the obtained transcription product, wherein the purification method is as follows:

(1) Adding RNase-Free H ₂ O to the transcript to make up 200. Mu.L;

(2) 200. Mu.L of a mixture A (water-saturated phenol: chloroform: isoamyl alcohol, v: v, 25:24:1) was added, vortexed for 10s, centrifuged at 13800 Xg for 5min at 4℃and then the upper water phase in the tube was transferred to a new tube;

(3) Adding an equal volume of mixed solution B (chloroform: isoamyl alcohol, v: v, 24:1) into a new tube, swirling for 10s, centrifuging at 4 ℃ and 13800 Xg for 5min, and then transferring the upper water phase in the tube into the new tube;

(4) Adding an equal volume of 5M ammonium acetate solution into a new tube, standing on ice for 15min after vortex mixing, centrifuging at 4 ℃ and 13800 Xg for 15min, and discarding the supernatant;

(5) RNA was cleaned by adding 70% ice-ethanol, then 70% ethanol was discarded, and resuspended in an appropriate amount of RNase-Free water (Solarbio, R1600) and quantified using the QubitTM RNA BR Assay Kit (Invitrogen, Q10211) kit.

5. And (3) performing mRNA capping reaction on the RNA transcription purification product obtained in the step (4), wherein the specific steps are as follows:

(1) RNA denaturation 60. Mu.g of the transcription purified product was incubated at 65℃for 15min for denaturation treatment and then transferred to ice.

(2) MRNA capping reaction, namely preparing a reaction system according to the formula shown in figure 7 after adding an RNA denaturation product, and incubating for 0.5h at 37 ℃ to obtain a capping product of which mRNA has a Cap 1 type structure, wherein Cap 1 is methyl guanosine.

And 6, purifying mRNA capping products, and synchronizing the step 4.

Mass analysis of mrna:

the synthetic mRNA is analyzed by adopting Qsep100 full-automatic nucleic acid protein analysis system and RNA CARTRIDGE KIT (C105110), and the specific steps are as follows:

(1) mRNA denaturation mRNA was dissolved in a1 Xsolution Buffer, denatured at 70℃for 2min, and immediately thereafter ice-cooled.

(2) Buffer and Buffer were prepared and P, W, C was placed in enzyme-free sterile water as described, S position Separation Buffer. The MC1 well was placed with a Low Marker of RNA.

(3) And (3) inserting and correcting the clamp, namely opening a clamp door above the instrument, inserting the clamp, and closing the clamp door. And after the Latch is clicked, the clamp is locked, the clamp is clicked Tool Recalibrate to correct again, the proper types of Voltage and ALIGNMENT MARKER are selected in the pop-up option boxes, the Start Calibration is clicked, a Calibration Succeed window is popped up when the correction is completed, and the click is confirmed.

(4) Program setting, namely clicking a blank cell below the Sample Position, popping up a 96-hole disc simulation diagram, selecting the Position of the placed Sample on the left side, double-clicking the Sample information input at the corresponding Position, and clicking OK after setting is completed. Clicking a blank box below the Method, popping up a test Method box, selecting proper ALIGNMENT MARKER and the Method, and clicking OK after completion. After the program is set, click Run and start electrophoresis separation.

(5) Data calculation analysis

As a result, the results are shown in FIG. 2, and the in vitro synthesized monkey pox mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000, A, mRNA-2949B and mRNA-2949C (shown in FIG. 2) bands were consistent with the target bands at concentrations of 1758 ng/. Mu.L, 1824 ng/. Mu.L, 2150 ng/. Mu.L, 1879 ng/. Mu.L, 2083 ng/. Mu.L and 1994 ng/. Mu.L, respectively.

3. Cell transfected protein expression detection of mRNA

1. Cell inoculation 293T cells (ATCC) were inoculated into 12 well plates, 3X 10 ⁵ cells per well, and transfected in a 37℃5% CO ₂ incubator until the cells reached 80-90% confluence.

2. The transfection complex is prepared by mixing 100. Mu.L of Opti-MEM+1. Mu.g mRNA with 2. Mu.L of TransIT-MRNA REAGENT and 2. Mu.L of TransIT-mRNA Boost, and then mixing and standing for 4min at room temperature.

3. And (3) transfecting cells, namely dripping the transfection complex into the cells and shaking up and down and left and right to uniformly distribute the transfection complex, incubating for 18 hours at 37 ℃ with 5% CO ₂, and collecting the cells without changing cell culture solution before and after transfection.

4. Total cell proteins were extracted after washing the cells twice with PBS, the cells were lysed by vortexing using cell lysate RIPA (Jin Pulai, P06M 11) +protease inhibitor 100× (Jin Pulai, P01C 01). After 30min in ice bath, the supernatant was collected by centrifugation at 13800 Xg for 15min at 4 ℃.

5. Quantification of total cellular proteins in the supernatant of lysis were quantified using BCA protein quantification kit (Jin Pulai, P06M 16). The cell lysis supernatant was mixed with BCA working solution, incubated at 37 ℃ for 45min, absorbance was measured at a562nm, and the total protein concentration of the cells was calculated.

6.Western blotting (WB) detection of target Protein expression Total Protein (10. Mu.g) separation was performed by Protein electrophoresis pre-Gel BoltTM to12%, bis-Tris,1.0mm, mini Protein Gel (Invitrogen, NW04120 BOX) electrophoresis (200V, 22 min). The isolated proteins on the gels were transferred onto iBlot 2 Transfer Stacks,PVDF (Invitrogen, IB 24001) membranes under gradient voltage (20 v,1min;23v,4min;25v,2 min), and then incubated at room temperature in 1×tbst with 5% nonfat milk powder, 20r/min, and blocked for 1h.

Anti-Monkeypox Virus/MPXV M1R Antibdy (Propox, SAA 0283), anti-Monkeypox Virus/MPXV B6R/SL-159 Polyclonal Antibody (Propox ,PVV13501)、Anti-MPXV-A29L rRmAb (Vazyme,RM3387)、Anti-MPXV-E8L rRmAb (Vazyme,RM3391)、Anti-MPXV-A35R rRmAb （Vazyme,RM3395）,20r/min, room temperature shaking 2H, washing membranes with 1 XTBST, 60R/min, shaking at room temperature for 10min, and repeating 3 times to completely remove primary Anti-remnants the secondary antibodies were diluted with horseradish peroxidase (HRP) labeled goat Anti-Mouse IgG secondary antibody (1:2000), HRP Goat Anti-Mouse IgG (H+L) (Abclone, AS 003) or horseradish peroxidase (HRP) labeled goat Anti-rabbit IgG secondary antibody (1:1000), (Biyun, A0208), 20R/min, shaking at room temperature for 1H, washing membranes with 1 XTBST, 60R/min, shaking at room temperature for 10min, and repeating 3 times to completely remove secondary Anti-remnants.

HRP-labeled antibody-bound antigen was detected in a chemiluminescent apparatus using ECL chemiluminescent hypersensitivity chromogenic kit (next holothurian, 36208ES 60) incubated with the membrane at room temperature for 3min in the absence of light. The expression of the antigen protein of interest was verified by alignment of the visualized bands with the protein marker, pageRulerTM Prestained Protein Ladder (Invitrogen, 26617) bands by exposure. Membrane regeneration solution (soribao, SW 3020) was used to remove antibodies on the membrane, and after the membrane was again blocked, reference antibodies were incubated to detect the uniformity of the protein loading amount. The antibody adopts beta-actin Rabbit monoclonal antibody diluent (1:50000), ACTB Rabbit mAb (Abclonal, AC 038), and the secondary antibody adopts goat anti-Rabbit IgG secondary antibody diluent marked by horseradish peroxidase (HRP) (1:10000), goat Anti-Rabbit IgG Secondary Antibody (HRP) (SSA 004, yiqiao Shenzhou).

Transfection and detection of mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000, A, mRNA-2949B and mRNA-2949C, respectively, were performed as described above, and the results are shown in FIG. 3. The results show that after HEK293T cells are transfected by in vitro synthesized monkey pox viruses mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000, A, mRNA-2949B and mRNA-2949C, the target antigens of the monkey pox viruses A29L, M1R, E L, A R and B6R are detected by WB.

4. Preparation of multi-antigen monkey pox mRNA vaccine

(1) Preparation of mRNA-2949-LNP, mRNA-3000-LNP, mRNA-2949A-LNP, mRNA-3000A-LNP, mRNA-2949B-LNP and mRNA-2949C-LNP lipid nanoparticle vaccine

The cationic lipid material (SM 102), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), cholesterol, and DMG-PEG2000 ethanol were completely dissolved. The lipid material ethanol solution is mixed with mRNA-2949, mRNA-3000, mRNA-2949A, mRNA-3000, mRNA-A, mRNA-2949B and 20mM sodium citrate buffer solution (pH 4.0) of mRNA-2949C according to the mol ratio of 50:10:38.5:1.5, and the mixture is mixed with the lipid material ethanol solution according to the volume ratio of 1:3 (lipid material: mRNA) at the flow rate of 12mL/min in a Micanana nano-drug preparation system. The collected sample was diluted 50-fold in DPBS buffer, concentrated by centrifugation and ultrafiltration at 4℃and 2000 Xg through a 50kDa PES ultrafiltration tube, and the ethanol content of the sample was removed. Finally, the vaccine formulation passing through the 0.22 μm filter was adjusted to the appropriate concentration with DPBS buffer for further experiments.

Example 2 detection of serum antibodies after immunization of mice with monkey poxvirus mRNA vaccine

1. Mouse muscle immunization of multi-antigen monkey poxvirus mRNA vaccine:

The experimental animals BALB/C mice (females, 6-8 weeks, 16-18g, beijing vellum) were randomly divided into monkey poxvirus mRNA-2949-LNP (mrna=25 μg/min), mRNA-3000-LNP (mrna=25 μg/min), mRNA-2949A-LNP (mrna=25 μg/min), mRNA-3000A-LNP (mrna=25 μg/min), mRNA-2949B-LNP (mrna=25 μg/min), mRNA-2949C-LNP (mrna=25 μg/min) and negative control group (5, normal feeding) and mice were immunized by intramuscular injection. Two immunizations were performed and sufficient mouse serum was obtained on day 10 post immunization using orbital bleeding.

2. Mouse muscle immunity and serum antibody detection of multi-antigen monkey poxvirus mRNA vaccine

The experimental animals BALB/C mice (females, 6-8 weeks, 16-18g, beijing velariwa) were randomly divided into different immunization schedule groups mRNA-2949-LNP (mrna=25 μg/min), mRNA-3000-LNP (mrna=25 μg/min), mRNA-2949A-LNP (mrna=25 μg/min), mRNA-3000A-LNP (mrna=25 μg/min), mRNA-2949B-LNP (mrna=25 μg/min), mRNA-2949C-LNP (mrna=25 μg/min) immune groups and negative control groups (PBS, ph=7.4) (5 per group, normal feeding), and mice were immunized by intramuscular injection. Two LNP-mRNA groups were immunized 14 days apart. Sufficient amounts of mouse serum were obtained on day 10 after each immunization using orbital bleeds. The in vivo immunopotentiation of the macaque poxvirus vaccine was evaluated by detecting the production of mouse immune serum monkey poxvirus antigen specific binding antibodies IgG by ELISA.

ELISA method for detecting generation of monkey pox virus antigen specific binding antibody IgG in immune mouse serum comprises the following steps:

(1) Coating A29L, A R, B6R, M1R, E L protein was diluted to 1 ng/. Mu.l with carbonate buffer (50 mM, pH 9.6,0.22 μm filter), respectively. 100 μl of protein diluent was added to each well of a 96-well plate, sealed, and left at 4deg.C overnight;

(2) Washing the plate, namely pouring a 96-well plate to remove protein coating liquid after coating overnight, adding 200 mu L of washing liquid (1 XTBS containing 0.2% Tween-20) into each well, gently shaking by hands for 30s, drying by beating on paper, and repeating for 6 times;

(3) Blocking 200. Mu.l of blocking solution (1 XTBS with 2% BSA) was added to each well and incubated for 2h at 37℃in an incubator;

(4) Plate washing, namely pouring a 96-well plate to remove the sealing liquid, adding 200 mu l of washing liquid (1 XTBS containing 0.2% Tween-20) into each well, gently shaking by hands for 30 seconds, and then drying on paper by beating, and repeating for 6 times;

(5) Incubating primary antibody, namely carrying out 10-time gradient dilution on immune mouse serum by using antibody diluent (washing liquid containing 0.5% BSA) to obtain serum with different dilutions from 10-1 to 10-6, adding 100 μl of serum diluent into each well, and incubating for 2 hours at 37 ℃ in an incubator;

(6) Plate washing, namely pouring a 96-well plate to remove serum diluent, adding 200 mu L of washing liquid (1 XTBS containing 0.2% Tween-20) into each well, gently shaking by hands for 30 seconds, and then drying on paper by beating, and repeating for 6 times;

(7) Incubating the secondary antibody, namely diluting HRP-conjugated Goat anti-Mouse IgG (H+L) (ABclonal, AS 003) with an antibody diluent (washing liquid containing 0.5% BSA) 5000 times to obtain a secondary antibody diluent, adding 100 mu L of the secondary antibody diluent into each hole, and incubating for 1H at a temperature of 37 ℃;

(8) Plate washing, namely pouring a 96-well plate to remove secondary antibody diluent, adding 200 mu l of washing liquid (1 XTBS containing 0.2% Tween-20) into each well, gently shaking by hands for 30 seconds, and then drying on paper in a beating mode, and repeating for 6 times;

(9) Color development, namely adding 100 mu lTMB substrate (Tiangen, RA 107) into each hole, and incubating for 20 min at room temperature in dark place;

(10) Terminating the color development, namely adding 50 mu L of 2M H ₂SO₄ into each hole, and detecting the OD value of A450 on an enzyme label instrument;

(11) And judging the IgG titer of the serum binding antibody, namely, the OD of one diluted serum is more than or equal to 2.1 with the OD of the negative control, the OD of the next dilution is less than 2.1 with the OD of the negative control, and the dilution is the corresponding antibody titer of the serum sample (calculated as 0.05 if the OD of the negative control is less than 0.05).

As shown in FIG. 4, monkey poxvirus mRNA vaccine mRNA-2949-LNP, mRNA-3000-LNP, mRNA-2949A-LNP, mRNA-3000A-LNP, mRNA-2949B-LNP and mRNA-2949C-LNP immune mouse serum all detected the production of high titers of antigen-specific binding antibody IgG, with A29L average binding antibody IgG titers of about 1:12800 (mRNA-2949-LNP group), 1:10400 (mRNA-3000-LNP group), 1:26000 (mRNA-2949A-LNP group), 1:18000 (mRNA-3000A-LNP group) 1:22000 (mRNA-2949B-LNP group) and 1:36000 (mRNA-2949C-LNP group); A35R average binding antibody IgG titres were about 1:14000 (mRNA-2949-LNP group), 1:44000 (mRNA-3000-LNP group), 1:28000 (mRNA-2949A-LNP group), 1:28000 (mRNA-3000A-LNP group) and 1:120000 (mRNA-2949B-LNP group), M1R average binding antibody IgG titres were about 1:64000 (mRNA-2949-LNP group), 1:80000 (mRNA-3000-LNP group), 1:660000 (mRNA-2949A-LNP group), 1:240000 (mRNA-3000A-LNP group), 1:280000 (mRNA-2949B-LNP group) and 1:52000 (mRNA-2949C-LNP group), B6R average binding antibody IgG titres were about 1:14000 (mRNA-2949-LNP group), 1:140000 (mRNA-3000-LNP group), 1:14400 (mRNA-2949-LNP group) and 1:14400 (mRNA-2949A-LNP group), 1:260000 (mRNA-3000A-LNP group), 1:84000 (mRNA-2949B-LNP group) and 1:140000 (mRNA-2949C-LNP group), E8L average binding antibody IgG titers of about 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) (as shown in FIG. 4).

3. Detection of neutralizing antibody of live virus of serum vaccinia Tiantan strain of immunized mice of monkey poxvirus mRNA vaccine

The neutralizing effect of the mouse immune serum on the poxvirus live virus was evaluated by the vaccinia Tiantan strain live virus, and the neutralizing antibody titer NT ₅₀ was determined, thereby evaluating the in vivo immune efficacy of the monkey poxvirus mRNA-2949-LNP, mRNA-3000-LNP, mRNA-2949A-LNP, mRNA-3000A-LNP, mRNA-2949B-LNP and mRNA-2949C-LNP vaccines.

Vaccinia virus neutralizing antibody NT ₅₀ potency assay:

The antibody neutralization effect of monkey pox virus mRNA vaccine immune mouse serum is evaluated by utilizing vaccinia Tiantan strain live virus. The vaccinia Tiantan strain live virus used for evaluation is newly provided by the national food and drug verification institute. The specific detection method comprises the following steps:

The mice immune serum was serially diluted 3-fold from 1/30 with DMEM complete medium, resulting in 6 different dilutions of serum incubated with virus at 37 ℃. And a virus-free cell control group and a virus control group of a serum-free sample are simultaneously arranged. After 1h incubation, cells were cultured in 2×10 ⁴ Vero cells, 37 ℃ and 5% co ₂ per well. Because the Tiantan strain live virus enters cells, firefly luciferase is expressed, and after 48 hours, the firefly luciferase reacts with a luminescent substrate and luminescence detection is carried out. The percentage of virus inhibition was calculated by comparison with the viral control luminescence value. The dilution factor of serum when pseudovirus was 50% inhibited can be calculated by the calculation formula, thereby calculating half inhibition dilution. Half inhibition dilutions are used to represent half neutralization dilutions NT ₅₀, the case of neutralizing activity of serum antibodies on viruses.

As shown in FIG. 5, the monkey pox virus mRNA-2949-LNP, mRNA-3000-LNP, mRNA-2949A-LNP, mRNA-3000A-LNP, mRNA-2949B-LNP and mRNA-2949C-LNP group vaccine immunized mouse serum has neutralization effect on the live vaccinia space plant virus. After two immunizations, the average virus-neutralizing antibody titer NT ₅₀ is mRNA-2949:2323, mRNA-3000:2185, mRNA-2949 A:1:5938, mRNA-3000 A:1:5042, mRNA-2949 B:1:5053, and mRNA-2949 C:1:6163, respectively.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the present invention.

Claims

1. An mRNA characterized in that the mRNA is as set forth in any one of A1) -A2) below:

A1 A mRNA sequence obtained by replacing T in SEQ ID No. 6 of the sequence Listing with U;

A2 A Cap structure is connected to the 5 'end of the mRNA of A1) and/or ployA structure is connected to the 3' end.

2. Use of the mRNA of claim 1 for the preparation of an mRNA vaccine for preventing infection by monkey poxvirus.

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

4. A biological material related to the mRNA of claim 1, wherein the biological material is any one of B1) -B6):

b1 A nucleic acid molecule encoding the mRNA of claim 1;

B2 An expression cassette comprising the nucleic acid molecule of B1);

B5 A recombinant bacterium comprising the nucleic acid molecule of B1), a recombinant bacterium comprising the expression cassette of B2), or a recombinant bacterium comprising the recombinant vector of B3).

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

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

7. An mRNA complex comprising a delivery vehicle comprising an ionizable lipid nanoparticle and the mRNA of claim 1.

8. The mRNA complex of claim 6, wherein 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 of claim 7, wherein the ionizable lipid nanoparticle is a cationic lipid nanoparticle.

10. The mRNA complex according to claim 7, wherein the mRNA complex is an ionizable lipid-mRNA lipid nanoparticle comprising an ionizable lipid, the mRNA of claim 1, a pegylated lipid, 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine, and cholesterol.

11. Use of the biomaterial of any one of claims 4-5 or the mRNA complex of any one of claims 6-10 for the preparation of an mRNA vaccine for the prevention of monkey pox virus.