Monkey pox virus multi-antigen monovalent vaccine and application thereof

Abstract

The invention provides a monkey pox virus multi-antigen monovalent vaccine and application thereof. Specifically, the invention provides a monkey pox virus monovalent multi-antigen fusion protein, and the fusion protein comprises a first fusion protein element containing A35R and M1R proteins and a second fusion protein element containing A29L and B6R proteins from an N terminal to a C terminal. According to the invention, A35R, M1R, A29L and B6R are subjected to reasonable antigen combination to design a protein molecule, and the protein molecule is prepared into a monovalent mRNA vaccine. Experimental results show that the mRNA vaccine provided by the invention can ensure high-efficiency expression of target protein in vivo and can induce a high-level neutralizing antibody.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically relates to a monkeypox virus multi-antigen monovalent vaccine and application thereof. Background Art

[0002] Monkeypox is a disease caused by infection with the monkeypox virus (MPXV). Monkeypox usually presents as a self-limiting infection, with symptoms including fever, headache, fatigue, swollen lymph nodes, and skin lesions. Monkeypox was first discovered in 1958, when two pox-like outbreaks occurred in a group of monkeys kept for research, hence the name "monkeypox". Currently, all monkeypox vaccines approved (for emergency use) worldwide are live virus vaccines developed for smallpox virus, which have clear side effects and are limited in the population that can be vaccinated. There are two main viral forms of monkeypox virus antigens: enveloped virus (EV) and mature virus (MV). The A35R protein from EV plays a role in the structure and function of the virus, and the B6R protein from EV is involved in the immune regulation and infection process of the virus. In addition, the M1 protein from MV is involved in the assembly of virus particles, and the A29 protein from MV plays a role in the adsorption of the virus and entry into host cells. Therefore, A29, M1, B6, and A35 were selected as key antigens for the design of monkeypox vaccines.

[0003] Currently, there is no monkeypox mRNA vaccine on the market. Conventional multi-antigen / multivalent mRNA vaccine preparation is to prepare each antigen separately through template linearization, in vitro transcription, purification and mixed encapsulation. Such a complex process means that as the number of antigens increases, the production investment will increase, the equipment usage will increase, and the production efficiency will decrease.

[0004] Therefore, there is an urgent need in the art to develop an MPXV multi-immunogen chimeric antigen and a corresponding mRNA vaccine that has a simple preparation process, high production capacity, low cost, and can effectively stimulate a specific immune response against monkeypox virus. Summary of the invention

[0005] The purpose of the present invention is to provide an mRNA immunogenic composition, structural features related to the mRNA immunogenic composition, mRNA-lipid complex, method for preparing mRNA vaccine, use in preparing drugs for preventing and / or treating monkeypox virus, methods for generating immune response against monkeypox virus, preventing monkeypox virus infection and treating. The monkeypox virus multi-antigen mRNA vaccine of the present invention based on the mRNA immunogenic composition can efficiently stimulate immune protection against monkeypox virus, thereby effectively preventing and / or treating monkeypox virus infection.

[0006] In the first aspect of the present invention, a monkeypox virus monovalent multi-antigen fusion protein is provided, wherein the fusion protein has a structure as shown in Formula Ia or Formula Ib from the N-terminus to the C-terminus: R1-L1-R2 (Formula Ia) R2-L1-R1 (Formula Ib) in, R1 is the first fusion protein element comprising A35R and M1R proteins from N-terminus to C-terminus; L1 is a key or joint element; R2 is a second fusion protein element comprising A29L and B6R proteins from N-terminus to C-terminus; “-” represents a peptide bond.

[0007] In another preferred embodiment, the monkeypox virus includes West African evolutionary branch clade I, West African evolutionary branch clade IIa, and West African evolutionary branch clade IIb.

[0008] In another preferred embodiment, the monkeypox virus is the West African evolutionary branch clade IIb.

[0009] In another preferred embodiment, the monkeypox virus includes A.1, B.1, A1.1, A.2, B1.10, B1.7, B1.2, and B1.3.

[0010] In another preferred embodiment, the monkeypox virus includes MPXV_USA_2022_MA001.

[0011] In another preferred embodiment, the first fusion protein element comprises from N-terminus to C-terminus: P1-A35R-linker1-M1R; Wherein, the P1 is a signal peptide, The A35R is the monkeypox virus A35R protein or an antigenic fragment thereof (or a derivative peptide thereof), The linker1 is a flexible peptide linker. The M1R is the monkeypox virus M1R protein or an antigenic fragment thereof (or a derivative peptide thereof).

[0012] In another preferred embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0013] In another preferred example, the amino acid sequence of P1 is shown in SEQ ID NO:1.

[0014] In another preferred example, the amino acid sequence of A35R comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:16, or the amino acid sequence from positions 98 to 181 as shown in SEQ ID NO:16.

[0015] In another preferred example, the amino acid sequence of A35R is the amino acid sequence from position 98 to position 181 as shown in SEQ ID NO:16.

[0016] In another preferred example, the amino acid sequence of M1R comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:17, or the amino acid sequence from position 1 to position 250 as shown in SEQ ID NO:17.

[0017] In another preferred example, the amino acid sequence of the M1R is the amino acid sequence from position 1 to position 250 as shown in SEQ ID NO:17.

[0018] In another preferred embodiment, the flexible peptide linker comprises 0-30 amino acids, preferably 1-10 amino acids.

[0019] In another preferred embodiment, the flexible peptide linker is 1-4 GGGGS and / or GGGS.

[0020] In another preferred embodiment, the linker1 is (G3S)4, and the sequence is shown in SEQ ID NO:3.

[0021] In another preferred embodiment, the second fusion protein element comprises from the N-terminus to the C-terminus: P2-A29L-linker2-B6R; Wherein, the P2 is a signal peptide, The A29L is the monkeypox virus A29L protein or an antigenic fragment thereof (or a peptide derived therefrom), The linker2 is a flexible peptide linker. The B6R is the monkeypox virus B6R protein or its antigenic fragment (or its derived peptide segment).

[0022] In another preferred example, the amino acid sequence of P2 is shown in SEQ ID NO:2.

[0023] In another preferred example, the amino acid sequence of A29L comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:18, or the amino acid sequence from positions 1 to 40 as shown in SEQ ID NO:18.

[0024] In another preferred example, the amino acid sequence of A29L is the amino acid sequence from position 1 to position 40 as shown in SEQ ID NO:18.

[0025] In another preferred example, the amino acid sequence of B6R comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:19, or the amino acid sequence from positions 20 to 317 as shown in SEQ ID NO:19.

[0026] In another preferred example, the amino acid sequence of B6R is the amino acid sequence from position 20 to position 317 as shown in SEQ ID NO:19.

[0027] In another preferred embodiment, the linker2 is (G3S)4, and the sequence is shown in SEQ ID NO:3.

[0028] In another preferred embodiment, the fusion protein comprises from the N-terminus to the C-terminus: P1-A35R-linker1-M1R-L1-P2-A29L-linker2-B6R; Wherein, P1 and P2 are each independently a signal peptide, The amino acid sequence of A35R is the amino acid sequence from position 98 to position 181 as shown in SEQ ID NO: 16, The amino acid sequence of the M1R is the amino acid sequence from position 1 to position 250 as shown in SEQ ID NO: 17, The amino acid sequence of A29L is the amino acid sequence from position 1 to position 40 as shown in SEQ ID NO: 18, The amino acid sequence of B6R is the amino acid sequence from position 20 to position 317 as shown in SEQ ID NO: 19, The linker1 and linker2 are each independently a flexible peptide linker, The L1 is a connector element.

[0029] In another preferred embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0030] In another preferred example, the amino acid sequence of P1 is shown in SEQ ID NO:1.

[0031] In another preferred example, the amino acid sequence of P2 is shown in SEQ ID NO:2.

[0032] In another preferred embodiment, the fusion protein has the activity of simultaneously binding to A35, M1, A29 and B6 monkeypox virus antigens.

[0033] In another preferred embodiment, the flexible peptide linker comprises 0-30 amino acids, preferably 1-10 amino acids.

[0034] In another preferred embodiment, the flexible peptide linker is 1-4 GGGGS and / or GGGS.

[0035] In another preferred embodiment, the linker1 is (G3S)4, and the sequence is shown in SEQ ID NO:3.

[0036] In another preferred embodiment, the linker2 is (G3S)4, and the sequence is shown in SEQ ID NO:3.

[0037] In another preferred example, the linker element is a 2A polypeptide or a derivative polypeptide thereof.

[0038] In another preferred example, the amino acid sequence of the linker element is shown in SEQ ID NO:4.

[0039] In another preferred embodiment, the fusion protein comprises from N-terminus to C-terminus: P1-A35R-(G3S)4M1R-2A-P2-A29L-(G3S)4-B6R, Among them, the A35R protein and the M1R protein are connected by (G3S)4 to form the first fusion protein element. A29L protein and B6R protein are connected by (G3S)4 to form the second fusion protein element. The first fusion protein and the second fusion protein are connected via a linker element 2A.

[0040] In another preferred example, the amino acid sequence of P1 is shown in SEQ ID NO:1.

[0041] In another preferred example, the amino acid sequence of P2 is shown in SEQ ID NO:2.

[0042] In another preferred example, the amino acid sequence of the fusion protein is shown in SEQ ID NO:5.

[0043] In the second aspect of the present invention, an isolated nucleotide is provided, wherein the nucleotide encodes the fusion protein described in the first aspect of the present invention.

[0044] In another preferred embodiment, the nucleotide has a structure of Formula II: Z1-Z2-Z3-Z4-Z5 (II) In the formula, Z1 is none or 5' capping element; Z2 is the 5′-UTR element; Z3 is a nucleotide sequence encoding the fusion protein described in the first aspect of the present invention; Z4 is the 3′-UTR element; Z5 is the polyA tail element; "-" means nothing or a connecting sequence.

[0045] In another preferred embodiment, the 5' capping element is selected from the group consisting of cap0 (m7G5'ppp5'Np), cap1 (m7G5'ppp5'NmpNp) and cap2 (m7G5'ppp5'NmpNmpNp); preferably cap1.

[0046] In another preferred embodiment, the nucleotide is mRNA.

[0047] In another preferred example, the nucleotide sequence is shown as SEQ ID NO:7.

[0048] In another preferred embodiment, the mRNA is encapsulated in liposomes to form liposome nanoparticles.

[0049] In another preferred embodiment, the average particle size of the liposome nanoparticles is 10-500 nm, preferably 60-100 nm, and more preferably 60-80 nm.

[0050] In another preferred embodiment, the liposome nanoparticles include a liposome layer located on the outside and the mRNA located on the inside.

[0051] In another preferred embodiment, the components of the liposome layer include any liposome layer, preferably selected from the following group: PEG-modified lipids (such as DMG-PEG 2000), neutral lipids (such as DSPC), cationic lipids (such as SM102), cholesterol, or a combination thereof.

[0052] In another preferred embodiment, the liposome layer of the nanoparticle comprises any liposome layer, preferably comprising cationic lipids, cholesterol, phosphatidylcholine and PEG-modified lipids.

[0053] In another preferred embodiment, the PEG-modified lipid is selected from the group consisting of DMG-PEG 2000, C14-PEG2000, C16-PEG2000, or a combination thereof.

[0054] In another preferred embodiment, the PEG-modified lipid is DMG-PEG 2000.

[0055] In another preferred embodiment, the cationic lipid is selected from the following group: SM102, DODAP, D-Lin-MC3-DMA, DODMA, or a combination thereof.

[0056] In another preferred embodiment, the cationic lipid is SM102.

[0057] In the third aspect of the present invention, a vector is provided, wherein the vector contains the nucleotide as described in the second aspect of the present invention.

[0058] In another preferred embodiment, the vector is: pET vector, pGEM-T vector, pcDNA3.1, or a combination thereof.

[0059] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector as described in the third aspect of the present invention, or the nucleotide as described in the second aspect of the present invention is integrated into the genome.

[0060] In a fifth aspect of the present invention, a pharmaceutical composition is provided, comprising the following components: (a) the fusion protein according to the first aspect of the present invention, or the nucleotide according to the second aspect of the present invention; and (b) a pharmaceutically acceptable carrier.

[0061] In another preferred embodiment, the content of component (a) is 0.1-99.9wt%, preferably 10-99.9wt%, and more preferably 70%-99.9wt%.

[0062] In another preferred embodiment, the dosage form of the pharmaceutical composition is liquid, solid, or gel.

[0063] In another preferred embodiment, the pharmaceutical composition is an mRNA vaccine composition.

[0064] In another preferred embodiment, the vaccine composition comprises the nucleotide (mRNA) as described in the second aspect of the present invention, and liposomes.

[0065] In another preferred embodiment, the vaccine composition is liposome nanoparticles.

[0066] In another preferred embodiment, the average particle size of the liposome nanoparticles is 10-500 nm, preferably 60-100 nm, and more preferably 60-80 nm.

[0067] In another preferred embodiment, the liposome nanoparticles include a liposome layer located on the outside and the mRNA located on the inside.

[0068] In another preferred embodiment, the components of the liposome layer include any liposome layer, preferably selected from the following group: PEG-modified lipids (such as DMG-PEG 2000), neutral lipids (such as DSPC), cationic lipids (such as SM102), cholesterol, or a combination thereof.

[0069] In another preferred embodiment, the liposome layer of the nanoparticle comprises any liposome layer, preferably comprising cationic lipids, cholesterol, phosphatidylcholine and PEG-modified lipids.

[0070] In another preferred embodiment, the PEG-modified lipid is selected from the group consisting of DMG-PEG 2000, C14-PEG2000, C16-PEG2000, or a combination thereof.

[0071] In another preferred embodiment, the PEG-modified lipid is DMG-PEG 2000.

[0072] In another preferred embodiment, the cationic lipid is selected from the following group: SM102, DODAP, D-Lin-MC3-DMA, DODMA, or a combination thereof.

[0073] In another preferred embodiment, the cationic lipid is SM102.

[0074] In another preferred embodiment, the vaccine composition is administered in a manner selected from the group consisting of subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal spraying and aerosol inhalation.

[0075] In another preferred embodiment, the mRNA itself in the vaccine composition can also serve as an adjuvant.

[0076] In another preferred embodiment, the subject of administration of the pharmaceutical composition or preparation includes humans or non-human animals.

[0077] In another preferred embodiment, the non-human animals include: rodents (such as rats, mice), primates (such as monkeys).

[0078] In another preferred embodiment, the pharmaceutical composition is used to prevent and / or treat monkeypox virus infection.

[0079] In another preferred embodiment, the prevention and / or treatment of monkeypox virus infection includes delaying the development of symptoms associated with monkeypox virus infection and / or reducing the severity of these symptoms.

[0080] In another preferred embodiment, the prevention and / or treatment of monkeypox virus infection also includes alleviating the symptoms associated with existing monkeypox virus infection and preventing the occurrence of other symptoms.

[0081] In another preferred embodiment, the pharmaceutical composition or preparation can be administered in combination with other antiviral or anti-inflammatory drugs.

[0082] In another preferred embodiment, the other antiviral or anti-inflammatory drugs for combined administration are selected from the following group: viral replication inhibitors, hormonal anti-inflammatory drugs, biological response modifiers, monoclonal antibodies, or a combination thereof.

[0083] In a sixth aspect of the present invention, a method for preparing the fusion protein according to the first aspect of the present invention is provided, comprising the following steps: (a) culturing the host cell according to the fourth aspect of the present invention under suitable conditions to obtain a culture containing the fusion protein; and (b) purifying and / or separating the culture obtained in step (a) to obtain the fusion protein targeting A35, M1, A29 and B6 monkeypox virus antigens.

[0084] In the seventh aspect of the present invention, there is provided a use of the fusion protein as described in the first aspect of the present invention, or the nucleotide as described in the second aspect of the present invention, or the pharmaceutical composition as described in the fifth aspect of the present invention, for preparing a medicament, a reagent, a detection plate or a kit; wherein the reagent, the detection plate or the kit is used for: detecting monkeypox virus in a sample; wherein the medicament is used for treating and / or preventing monkeypox virus infection.

[0085] In another preferred embodiment, the agent is used to block the monkeypox virus from invading the human body.

[0086] In another preferred embodiment, the reagent is one or more reagents selected from the following group: isotope tracers, contrast agents, flow cytometry detection reagents, cell immunofluorescence detection reagents, nanomagnetic particles and imaging agents.

[0087] In another preferred embodiment, the reagent for detecting monkeypox virus in a sample is a contrast agent for detecting monkeypox virus (in vivo).

[0088] In another preferred embodiment, the detection is in vivo detection or in vitro detection.

[0089] In another preferred embodiment, the detection includes flow cytometry detection, cell immunofluorescence detection, or a combination thereof.

[0090] In another preferred embodiment, the monkeypox virus or its related disease is selected from the following group: monkeypox virus infection, other poxvirus (such as vaccinia virus) infection, or a combination thereof.

[0091] In another preferred embodiment, the monkeypox virus includes West African evolutionary branch clade I, West African evolutionary branch clade IIa, and West African evolutionary branch clade IIb.

[0092] In another preferred embodiment, the monkeypox virus includes A.1, B.1, A1.1, A.2, B1.10, B1.7, B1.2, and B1.3.

[0093] In the eighth aspect of the present invention, a method for generating an immune response against monkeypox virus is provided, comprising the steps of administering the fusion protein described in the first aspect of the present invention or the nucleotide described in the second aspect of the present invention or the pharmaceutical composition described in the fifth aspect of the present invention to a subject in need thereof.

[0094] In another preferred embodiment, the method comprises administering liposome nanoparticles containing the mRNA to a subject in need thereof.

[0095] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0096] In another preferred embodiment, the non-human mammal includes non-human primates (such as monkeys).

[0097] In another preferred embodiment, the method induces the production of neutralizing antibodies against monkeypox virus in the subject.

[0098] In the ninth aspect of the present invention, a method for preventing and / or treating monkeypox virus infection is provided, comprising the steps of administering to a subject in need thereof the fusion protein as described in the first aspect of the present invention, the nucleotide as described in the second aspect of the present invention, or the pharmaceutical composition as described in the fifth aspect of the present invention.

[0099] In another preferred embodiment, the method comprises administering liposome nanoparticles containing the mRNA to a subject in need thereof.

[0100] In another preferred embodiment, the prevention and / or treatment of monkeypox virus infection includes blocking the invasion of monkeypox virus into the human body.

[0101] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1The schematic diagram of the candidate mRNA sequence structure and the optimization result diagram in the embodiment of the present invention are shown. Among them, A is a diagram showing the effect of A35R and M1R connected by 2A or antigen protein B6R and M1R connected by 2A, and the results of the two on the expression of M1R are detected. The results show that the expression of M1R corresponding to A35R in front of M1R is higher than that of B6R in front of M1R; B is a comparison result of B6R expression corresponding to three signal peptides, among which B6R sig is the signal peptide of B6R itself, mouse immunoglobulin heavy chain sig is the signal peptide of mouse immunoglobulin heavy chain, tPA Sig is the signal peptide of human tissue plasminogen activator. The results show that the B6R expression level of mouse immunoglobulin heavy chain sig is the highest, so the signal peptide is selected as the signal peptide of A29L-(G3S)4-B6R fusion protein; C and D respectively represent the comparison results of the expression levels of A35R and M1R in the screening of A35R(98-181aa)-connector peptide-M1R by selecting two signal peptides, wherein mouse immunoglobulin heavy chain sig is the mouse immunoglobulin heavy chain signal peptide, mIL-21R sig is the mouse interleukin 21 receptor signal peptide, and the results show that the A35R(98-181aa)-connector peptide-M1R expression level using mIL-21Rsig is the highest, so the signal peptide is selected as the signal peptide of A35R(98-181aa)-connector peptide-M1R fusion protein, and A35R(98-181aa) indicates that the A35 sequence is relative to SEQ ID NO: The amino acid sequence from position 98 to position 181 of the sequence shown in 16; E is a schematic diagram of the candidate mRNA sequence structure, MPXV1 represents the mRNA sequence structure connected in sequence by a connecting chain: A35R-(G3S)4-M1R-2A-A29L-(G3S)4-B6R; MPXV2 represents a mixed mRNA sequence of A35R, M1R, A29L and B6R.

[0103] Figure 2 The results of protein immunoblotting of monkeypox vaccine antigen mRNA expression at the in vitro cellular level are shown. Among them, MPXV1 represents the mRNA sequence structure connected in sequence by a linker: A35R-(G3S)4-M1R-2A-A29L-(G3S)4-B6R; MPXV2 represents a mixed mRNA sequence of A35R, M1R, A29L and B6R.

[0104] Figure 3 The flowchart of mouse immunization and blood collection and the result graph of detecting humoral immune response of mice induced by mRNA vaccine are shown, among which A is the flowchart of mouse immunization and blood collection, and B is the result graph of antibody titer detection of serum collected on the 28th day of immunization and diluted at different multiples.

[0105] Figure 4The figure shows the results of enzyme-linked immunosorbent assay (ELISA) to measure the humoral immune response of mice induced by mRNA vaccine. "*" indicates comparison with the PBS control group in the same time period, the horizontal line indicates pairwise comparison, "*" indicates P<0.05; "**" indicates P<0.01, and "***" indicates P<0.001.

[0106] Figure 5 The figure shows the results of the monkeypox virus neutralizing antibody assay, and "*" indicates P < 0.05 compared with the control group PBS. DETAILED DESCRIPTION

[0107] Through a large number of experimental screenings, the present invention selects four highly immunogenic monkeypox virus surface proteins (A35R, M1R, A29L and B6R) as candidate target proteins for the first time, and transforms the proteins and screens the arrangement order and signal peptides, and finally obtains a monkeypox virus mRNA vaccine formed by sequentially connecting A35R, M1R, A29L and B6R monkeypox surface proteins or their antigenic fragments. The design method of the present invention can overcome the low expression efficiency of the target protein in the host cell and enhance its immunogenicity. The experimental results show that the mRNA vaccine of the present invention can ensure the efficient expression of the target protein in vivo and can induce a very high level of neutralizing antibodies. On this basis, the present invention is completed.

[0108] the term As used herein, the terms "fusion protein of the present invention", "monkeypox virus monovalent antigen fusion protein", "monkeypox virus multi-antigen monovalent vaccine", "mRNA vaccine", "MPXV1", and "mRNA vaccine of the present invention" are used interchangeably and refer to a monkeypox virus monovalent antigen fusion protein having the structure of Formula Ia or Formula Ib shown in the first aspect of the present invention.

[0109] As used herein, the terms "nucleotides of the present invention", "nucleotides encoding fusion proteins", "mRNA of the present invention", "mRNA for preventing monkeypox virus of the present invention", "mRNA molecules of the present invention", and "mRNA sequences of the present invention" are used interchangeably and refer to nucleotides encoding the fusion protein described in the first aspect of the present invention.

[0110] Monkeypox virus Monkeypox virus is a zoonotic virus belonging to the genus Orthopoxvirus. It is a double-stranded DNA virus similar to smallpox (VARV), cowpox (CPX) and vaccinia (VACV) viruses. MPV is oval in shape with a lipoprotein membrane on the outside. The genome is about 190 kb. Monkeypox can be divided into two different branches, Central Africa (also known as the Congo Basin) and West Africa, with high sequence similarity between the two branches. The West African branch is responsible for the widespread global spread of the current epidemic.

[0111] Clinical manifestations of infection with monkeypox virus are similar to those of smallpox, but the rash is milder and the mortality rate is lower. The virus can be transmitted between animals and humans through direct contact with lesions or body fluids. The virus was first isolated from monkeys and was named monkeypox virus. The main carriers of the virus are rodents.

[0112] Monkeypox virus monovalent antigen fusion protein of the present invention The present invention provides a fusion protein or a conjugate thereof that simultaneously binds to A35, M1, A29 and B6 monkeypox virus antigens.

[0113] Preferably, the fusion protein comprises from N-terminus to C-terminus: P1-A35R-linker1-M1R-L1-P2-A29L-linker2-B6R; Wherein, P1 and P2 are each independently a signal peptide, The amino acid sequence of A35R is the amino acid sequence from position 98 to position 181 as shown in SEQ ID NO: 16, The amino acid sequence of the M1R is the amino acid sequence from position 1 to position 250 as shown in SEQ ID NO: 17, The amino acid sequence of A29L is the amino acid sequence from position 1 to position 40 as shown in SEQ ID NO: 18, The amino acid sequence of B6R is the amino acid sequence from position 20 to position 317 as shown in SEQ ID NO: 19, The linker1 and linker2 are each independently a flexible peptide linker, The L1 is a connector element.

[0114] Preferably, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0115] Preferably, the amino acid sequence of P1 is as shown in SEQ ID NO:1.

[0116] Preferably, the amino acid sequence of P2 is as shown in SEQ ID NO:2.

[0117] Preferably, the fusion protein of the present invention has an amino acid sequence as shown in SEQ ID NO:5.

[0118] As used herein, the term "fusion protein" also includes variant forms having A35, M1, A29 and B6 monkeypox virus antigen binding activity. These variant forms include (but are not limited to): 1-3 (usually 1-2, preferably 1) amino acid deletion, insertion and / or substitution, adding or deleting one or several (usually within 3, preferably within 2, more preferably within 1) amino acids at the C-terminus and / or N-terminus, or adding amino acid fragments with smaller amino acid side chains as linkers (such as glycine, serine, etc.) at the N-terminus or C-terminus of small proteins. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus usually does not change the structure and function of the protein. In addition, the term also includes polypeptides of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0119] The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having substitution groups in one or more amino acid residues, or (iii) polypeptides formed by fusion of a polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (fusion proteins formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0120] A preferred class of active derivatives refers to polypeptides formed by replacing at most 5, preferably at most 3, and more preferably at most 1 amino acid with similar or similar properties compared to the amino acid sequence of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.

[0121] Table 1

[0122] The present invention also provides analogs of the fusion protein of the present invention. The difference between these analogs and the polypeptide of the present invention may be a difference in amino acid sequence, or a difference in modification form that does not affect the sequence, or both. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

[0123] The term "polynucleotide of the present invention" may include a polynucleotide encoding the fusion protein of the present invention, or may include additional coding and / or non-coding sequences.

[0124] The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or fusion proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the ultra-high affinity small protein or fusion protein encoded by it that targets the S protein.

[0125] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%.

[0126] mRNA vaccines The present invention also provides an mRNA vaccine for preventing monkeypox virus and a preparation method thereof. The monkeypox virus mRNA vaccine of the present invention has the advantages of relatively long expression time, strong immunogenicity, good protection, etc.

[0127] Typically, the method of the present invention includes: first obtaining the expression gene (or coding sequence) of the antigen protein by the PCR method, then obtaining the corresponding mRNA by in vitro transcription technology, and finally obtaining a highly effective mRNA vaccine.

[0128] mRNA vaccine is an in vitro prepared mRNA with expression activity, and its main structure includes 5' and 3' UTR and an open reading frame containing the expressed antigen. Compared with DNA vaccines, it does not require any nuclear localization signal and has no risk of integration into the genome. The mRNA vaccine encapsulated by liposome nanoparticles does not require any other adjuvants for assistance. The liposome nanoparticles and RNA themselves can act as adjuvants, and they can simultaneously activate the MHCI and MHCII presentation pathways, thereby greatly improving the immunogenicity of the vaccine.

[0129] In order to increase the expression of mRNA vaccines, a series of nucleic acid elements that promote mRNA expression are selected in the present invention. The elements include (but are not limited to): 5'UTR, signal peptide, antigen sequence and 3'UTR.

[0130] In the present invention, the preferred monkeypox virus proteins are A35R, M1R, A29L and B6R in sequence. The mRNA vaccine of the present invention comprises the mRNA encoding these monkeypox virus proteins in series.

[0131] In the present invention, SEQ ID NOs: 12-15 are the mRNA sequences of monkeypox virus A35R, M1R, A29L and B6R proteins, respectively.

[0132] In a preferred embodiment, the mRNA vaccine of the present invention includes mRNA of monkeypox virus A35R, M1R, A29L and B6R proteins.

[0133] In the present invention, the monkeypox virus includes West African evolutionary branch clade I, West African evolutionary branch clade IIa, and West African evolutionary branch clade IIb.

[0134] In a preferred embodiment, the monkeypox virus includes A.1, B.1, A1.1, A.2, B1.10, B1.7, B1.2, and B1.3.

[0135] In a preferred embodiment, the monkeypox virus comprises MPXV_USA_2022_MA001.

[0136] In a preferred embodiment, the present invention selects the monkeypox virus strain (MPXV_USA_2022_MA001) on NCBI as the reference sequence because the sequence best matches the current reality of the monkeypox epidemic and is expected to play a greater role in controlling the epidemic.

[0137] In the present invention, four monkeypox virus proteins were selected as target proteins. The monkeypox virus strain (MPXV_USA_2022_MA001 (Genbank NO: 563414.2)) derived from the 2022 epidemic strain was used as the reference genome, and its protein names were named A35R, M1R, A29L, and B6R respectively.

[0138] The above protein sequence is designed by modifying the original sequence of monkeypox virus to overcome many defects and achieve the maximum effect of vaccine-induced immune response. The modified nucleic acid sequence is shown in SEQ ID NO:6.

[0139] In addition, the present invention compares multiple vaccine regimens, including four monovalent regimens, two bivalent regimens, and one quadrivalent regimen.

[0140] In the monovalent regimen, a vaccine was designed for each protein (A35R, M1R, A29L, B6R) as a target protein.

[0141] In the two-valent regimen, two proteins are selected in combination: A35R and M1R; or A29L and B6R.

[0142] In the quadrivalent regimen, four proteins are selected in combination: A35R, M1R, A29L, and B6R.

[0143] The vaccine design scheme of the present invention is different from the prior art method of combining protein vaccines, and the present invention is a monovalent multi-antigen vaccine.

[0144] In a preferred example, the amino acid sequence of the monkeypox virus vaccine of the present invention is shown in SEQ ID NO:5.

[0145] In addition, the present invention is modified by the following steps to overcome the problem of low expression efficiency of the target protein in the host cell and enhance its immunogenicity. The steps are as follows:

[0146] 1) Introduce signal peptide into expression plasmid; 2) Remove the transmembrane region; 3) Introduce polyA into the expression plasmid; In order to improve the expression efficiency and expression amount of the target protein in the host cells and induce a stronger immune response, a signal peptide sequence (SEQ ID NO: 1) was added to the N-terminus of the A35R and M1R fusion proteins; a signal peptide sequence (SEQ ID NO: 2) was added to the N-terminus of the A29L and B6R fusion proteins to enhance their immunogenicity.

[0147] The present invention also includes related products produced using the designed monovalent multi-antigen vaccine, including purified antigen proteins, polyclonal and monoclonal antibody reagents of monkeypox antigens, and related drugs.

[0148] The vaccine of the present invention induces specific neutralizing antibodies with high affinity to the immunogen in mice. The present invention also includes the preparation method of the relevant specific antigens and the induced neutralizing antibodies, and the variable region gene sequence design scheme based on these high affinity neutralizing antibodies.

[0149] Compositions and methods of administration The present invention also provides a composition, which comprises the liposome nanoparticles of the present invention and the mRNA vaccine of the present invention.

[0150] The compositions of the present invention include pharmaceutical compositions and vaccine compositions.

[0151] The composition of the present invention may contain nucleotides encoding multiple antigenic proteins (a combination of two or more of A35R, M1R, A29L and B6R, or a combination of four proteins of A35R, M1R, A29L and B6R).

[0152] The pharmaceutical composition or vaccine composition of the present invention can be prepared into various dosage forms, including (but not limited to): injection, suspension, spray, etc.

[0153] The main advantages of the present invention include: (1) The present invention provides a multi-immunogen fusion antigen against monkeypox virus, which comprises four immunogens in sequence: monkeypox virus A35R protein or its antigenic fragment (or its derivative peptide), monkeypox virus M1R protein or its antigenic fragment (or its derivative peptide), monkeypox virus A29L protein or its antigenic fragment (or its derivative peptide), and monkeypox virus B6R protein or its antigenic fragment (or its derivative peptide); wherein A35R and B6R are antigens specific to mature virus particles (MV) in cells, and M1R and A29L are antigens specific to extracellular enveloped virus particles (EV); a vaccine comprising these four immunogens can stimulate an immune response against two infectious virus particles.

[0154] (2) The present invention forms a monovalent multi-antigen by directly connecting multiple monkeypox antigen proteins or their antigenic fragments in series or connecting them in series through an appropriate linker sequence. The resulting monovalent multi-antigen not only retains the immunogenicity of each of the four immunogens, but also can more efficiently activate specific protective antibodies against monkeypox virus.

[0155] (3) The present invention is based on the latest reference genome of the monkeypox virus strain (MPXV_USA_2022_MA001 (Genbank NO: 563414.2)) on NCBI as the original sequence, and selects a protein with strong immunogenicity in the surface protein of the monkeypox virus as a candidate target protein to develop a vaccine. This sequence is completely different from the reference sequence and modification method used in the design of the vaccine target protein sequence in the prior art. The present invention designs a monovalent multi-antigen vaccine, which induces an immune response in the body through different combinations of target proteins, obtains high-titer antibodies that can block the infection of the monkeypox virus, and enhances the immune effect of the vaccine.

[0156] (4) The present invention designs a monovalent multi-antigen vaccine, detects the effects of specific immunity induced by different target proteins and their combinations, obtains the immunogen combination with the best protective effect, and obtains high-titer antibodies that can block viral infection.

[0157] (5) The present invention designs and modifies the target protein by removing the transmembrane region of the target protein and introducing signal peptide sequences in the original sequence, thereby improving the stability and efficiency of protein expression of the recombinant plasmid and ensuring that the target protein can be efficiently expressed in vivo.

[0158] (6) The present invention utilizes one mRNA to encode multiple protective antigens of the monkeypox virus MPXV, which can produce a high level of immune protection against the monkeypox virus and can be used to prepare vaccines or drugs for the prevention and treatment of monkeypox. The mRNA vaccine composition prepared by the present invention can be used for vaccination of the general population when needed, which is safer and therefore has broad application prospects in clinical practice.

[0159] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as the conditions described in the American Sambrook.J et al. Molecular Cloning Laboratory Guide (Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0160] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial channels.

[0161] The preparation method of the monkeypox monovalent vaccine provided by the present invention mainly comprises an amino acid sequence, a DNA sequence and an mRNA sequence. The amino acid sequence mainly refers to the antigen selected in the antigen design, preferably A35R (98-181aa, i.e., positions 98-181 of the amino acid sequence shown in SEQ ID NO: 16), M1R (1-250aa, i.e., positions 1-250 of the amino acid sequence shown in SEQ ID NO: 17), A29L (1-40aa, i.e., positions 1-40 of the amino acid sequence shown in SEQ ID NO: 18), and B6R (20-317aa, i.e., positions 20-317 of the amino acid sequence shown in SEQ ID NO: 19). The four antigens are combined and connected, and the linker is preferably (G3S)4 (sequence as shown in SEQ ID NO: 3) and a 2A peptide sequence (sequence as shown in SEQ ID NO: 4). NO:4), firstly, two antigens (A35R and M1R, or A29L and B6R) are fused with (G3S)4, and the four antigens correspond to two groups of fusions, namely, fusion protein A35R-(G3S)4-M1R and fusion protein A29L-(G3S)44-B6R; then, the two groups of fusion antigens are connected with the 2A sequence to obtain a protein amino acid chain MPXCV1. The preferred connection sequence of the present invention is A35R-(G3S)4-M1R-2A-A29L-(G3S)4-B6R.

[0162] Example 1 Preparation of Monkeypox Virus mRNA (1) Design of amino acid sequence of candidate antigen fusion protein and plasmid construction The amino acid sequences of the A35R, M1R, A29L, and B6R proteins selected in the present invention are derived from the 2022 epidemic strain of monkeypox MPXV_USA_2022_MA001 (Genbank NO: 563414.2), among which the full-length amino acid sequences of the A35R, M1R, A29L, and B6R proteins are shown in SEQ ID NOs: 16-19, respectively.

[0163] First, the inventors optimized the sequential arrangement of A35R, M1R, A29L, and B6R proteins, using 2A to connect A35R and M1R to form a fusion protein: A35R-2A-M1R; using 2A to connect B6R and M1R to form a fusion protein: B6R-2A-M1R, and compared the expression levels of M1R in the two.

[0164] Western Blot results Figure 1 As shown in Figure A, compared with B6R placed in front of M1R, A35R placed in front of M1R makes the expression level of M1R higher. Therefore, the present invention puts the amino acid sequence of A35R in the first place, and through screening, the final selection sequence is placed at the first place of the candidate antigen fusion protein of the present invention, such as the amino acid sequence of positions 98-181 in the A35R amino acid sequence shown in SEQ ID NO: 16 (abbreviated as A35R (98-181)).

[0165] Secondly, in the screening of B6R, three signal peptides were selected for comparison of expression levels, namely, the signal peptide of B6R itself (B6R sig), the signal peptide of immunoglobulin heavy chain from mice, and the signal peptide of human tissue plasminogen activator (tPA sig). Among them, the expression level of B6R using the signal peptide of mouse immunoglobulin heavy chain was the highest ( Figure 1 Therefore, this signal peptide was selected as the signal peptide of A29L-(G3S)4-B6R fusion protein.

[0166] In the screening of A35R(98-181)-(G3S)4-M1R, two signal peptides were selected for comparison of expression levels, namely, the immunoglobulin heavy chain signal peptide from mice (mouse immunoglobulin heavy chain signal peptide) and the interleukin 21 receptor signal peptide from mice (mIL-21R sig). A35R ( Figure 1 C) and M1R ( Figure 1The expression levels of A35R and M1R were higher when mIL-21R sig was used than when mouse immunoglobulin heavy chain signal peptide was used. Therefore, this signal peptide was selected as the signal peptide of A35R(98-181)-(G3S)4-M1R fusion protein.

[0167] The amino acid sequence of the candidate antigen fusion protein of the present invention is preferably arranged in the order of A35R-M1R-A29L-B6R from N-terminus to C-terminus, wherein the A35 signal peptide is preferably mIL-21R sig, (sequence as shown in SEQ ID NO: 1), fused to the N-terminus. The A29L signal peptide is preferably a mouse immunoglobulin heavy chain signal peptide, (sequence as shown in SEQ ID NO: 2). The connecting peptide between A35R and M1R and the connecting peptide between A29L and B6R are preferably (G3S) 4 (sequence as shown in SEQ ID NO: 3). The connecting peptide between A29L and B6R is preferably a 2A peptide sequence, (sequence as shown in SEQ ID NO: 4).

[0168] The sequence of the antigen fusion protein is shown in SEQ ID NO: 5. SEQ ID NO: 5 is optimized by human codons to obtain a nucleic acid sequence (sequence shown in SEQ ID NO: 6), and the nucleic acid sequence is constructed into a vector, and the upstream of SEQ ID NO: 6 contains a 5'UTR sequence (including a Kozak sequence), and the downstream contains a stop codon TGATGA sequence, a 3'UTR sequence and a PolyA sequence and a BsaI restriction site, and a candidate target mRNA sequence is obtained, named MPXV1 (sequence shown in SEQ ID NO: 7), and is constructed into a basic plasmid pUC57 (containing a T7 promoter) by conventional molecular biological methods to obtain a circular plasmid that can be used for in vitro transcription of mRNA. In addition, the protein sequences of A35R, M1R, A29L, and B6R derived from the 2022 epidemic strain of monkeypox MPXV_USA_2022_MA001 (Genbank NO: 563414.2) are respectively constructed into the vector, and the signal peptides of M1R and B6R are preferably mouse immunoglobulin heavy chain signal peptides. The amino acid sequences of the A35R, M1R, A29L, and B6R proteins modified with signal peptides are shown in SEQ ID NOs: 8-11, and the corresponding nucleic acid sequences are shown in SEQ ID NOs: 12-15. The sample in which the four mRNAs A35R, M1R, A29L, and B6R were mixed and encapsulated was named MPXV2. The schematic diagram of the candidate mRNA sequence structure is shown in Figure 1 Figure E in .

[0169] (2) Plasmid extraction After the plasmid construction is completed and the sequence is confirmed to be correct, the strain is preserved. The strain is inoculated in 2YT medium, kanamycin is added to the medium, and the bacteria are inoculated at a ratio of 200 ml of medium to 100 μL of bacterial solution. The shaker is cultured overnight at 37°C for 14-16 hours. When the OD600 is 2.0-3.0, the bacteria are collected by centrifugation. P1 buffer (25 mM Tris-HCl (pH 8.0), 10 mM EDTA, 50 mM glucose) is added to resuspend the bacteria, and then P2 buffer (250 mM NaOH, 1% (w / v) SDS (sodium dodecyl sulfate)) is added for lysis. Finally, P3 buffer (3M potassium acetate, 5M acetic acid) is added for neutralization, and then centrifuged to obtain the supernatant for Bestarose 6FF gel filtration. Finally, the sample is transferred to a 100KD ultrafiltration tube for concentration. After concentration, the sample is pipetted into a new collection tube, the concentration is measured and stored.

[0170] (3) Preparation of linearized plasmid template The supercoiled plasmid was digested with enzymes, and the 50 μL plasmid linearization system was as shown in Table 2: Table 2

[0171] Add each component to a centrifuge tube, mix well, and place in an incubator at 37±0.5℃ for 16 hours. After the reaction is completed, take a sample for agarose gel electrophoresis to confirm the linearization effect. Among them, the electrophoresis result of MPXV1 is a single band with the correct size. The completely linearized plasmid is recovered by isopropanol precipitation, and the recovered linearized plasmid is tested for RNase residues. The linearized plasmid without obvious degradation is used for the next step

[0172] (4) In vitro transcription The co-transcription capping in vitro transcription system is shown in Table 3 below: Table 3

[0173] After the co-transcription capping in vitro transcription system is mixed, it is placed in an incubator at 37℃±0.5℃ for 3 h. After the transcription reaction is completed, the template is digested with DNase I. After the transcription is completed, DNase I, GMP grade (2U / μL) is added and mixed, and then reacted at 37±0.5℃ for 30 min. Take out 1μL of sample, dilute it 10 times, and load it for agarose gel electrophoresis to observe whether the IVT template is completely digested. Then use OligodT column to purify to remove impurities such as protein and salt. Gel electrophoresis and Qsep analysis confirm that the purity of the obtained mRNA is >90% and there is no obvious degradation.

[0174] Example 2 mRNA in vitro cell transfection experiment (1) mRNA transfection 293T cells were cultured in 75 cm 2 The culture medium was DMEM supplemented with 10% fetal bovine serum (FBS). When the cells grew to 70–90% confluence, the cells were digested with trypsin to prepare a cell suspension with a cell suspension density of 2.5x10 5 , the cells were plated on a 24-well plate, 500 μL per well, and cultured overnight. The next day, the original culture medium was discarded before transfection and replaced with Opti-MEM™I reduced serum medium. 450 μl Opti-MEM™I culture medium was slowly added to each well of cells along the side wall. Prepare 2 Tube tubes and dilute the transfection reagent and mRNA sample respectively using Opti-MEM™I culture medium. Add 1.5 μL transfection reagent Lipofectamin mRNA Max (Thermo Fisher Scientific) to 25 μL Opti-MEM™ I culture medium, and add 2 μg of the mRNA stock solution prepared in Example 1 to 25 μL Opti-MEM™ I culture medium, wherein A35R, M1R, A29L, and B6R of MPXV2 were mixed in a mass ratio of 1:1:1:1. Mix the mixed solutions in the two tubes by blowing and mixing them separately and let them stand for 10 minutes. After the waiting time is over, mix the above transfection reagent with the mRNA stock solution and let it stand for 5 minutes. Slowly drip 50 μL of the mRNA-transfection reagent mixed liquid into the culture plate, mix well, and culture at 37°C for 24 h.

[0175] (2) Western blotting The cell supernatant in the culture well plate in step (1) was discarded, 100 μl of 5× loading buffer was added to each well, and universal nuclease was added, lysed at room temperature for 5 min, and transferred to a 0.5 ml EP tube. 8-20% SDS-PAGE gel electrophoresis was used for separation; after separation, the membrane was transferred to transfer the protein to the PVDF membrane; then, the PVDF membrane was blocked in 3% bovine serum albumin, and mouse serum immunized with A35R, M1R, A29L, and B6R antigen proteins was used as the primary antibody, and goat anti-mouse IgG-HRP was used as the secondary antibody, and the PVDF membrane was incubated for 1 hour each; finally, the color was developed using a color developing solution.

[0176] The results are as follows Figure 2 As shown, Figure 2 The expression of each antigen mRNA in the cell is shown; Figure 2It can be seen that A35R, M1R, A29L, and B6R in MPXV1 and MPXV2 are expressed in cells, and the target bands can be detected. Among them, A35R and M1R in MPXV1 are fused into one molecule with a theoretical molecular weight of 39.9 kDa, and A29L and B6R are fused into one molecule with a theoretical molecular weight of 38.1kDa; the four proteins of MPXV1 are expressed separately, with the theoretical molecular weight of A35R being 20.1 kDa, the theoretical molecular weight of M1R being 27.3kDa, the theoretical molecular weight of A29L being 12.6kDa, and the theoretical molecular weight of B6R being 33.1kDa. The apparent molecular weight in the protein immunoblotting experiment is slightly higher than the theoretical molecular weight, which is due to post-translational modification.

[0177] Example 3 Lipid nanoparticle (LNP) packaging of mRNA Cationic lipid (SM102, Catalog No. 2089251-47-6, Sinobond Biotech Co., Ltd.), cholesterol (Catalog No. C3045-5G, Sigma-Aldrich Trading Co., Ltd.), phosphatidylcholine (DSPC, Catalog No. 5653PNA178, Avantor Life Sciences Co., Ltd.), and PEG lipid (DMG-PEG2000, Catalog No. 002005, Avantor Life Sciences Co., Ltd.) were mixed in a ratio of 50:38.5:10:1.5, and then the above mRNA was encapsulated in liposomes using a rapid nanodrug preparation instrument (Mianna (Shanghai) Instrument Technology Co., Ltd., INano L / 202101L24), in which A35R, M1R, A29L, and B6R of MPXV2 were encapsulated in a mass ratio of 1:1:1:1. After encapsulation, the buffer solution was replaced with PBS by dialysis, and then concentrated using a 50 KD ultrafiltration concentration tube. The mRNA-LNP was concentrated to a concentration of about 0.2 mg / ml. The final product that passed the quality inspection was stored at 4°C until use. The quality inspection results are shown in Table 4:

[0178] Table 4

[0179] Example 4 Experiment on mRNA vaccine immunization of mice In this example, female mice of BALB / c strain aged 6-8 weeks were used for animal experiments; the experimental groups were divided into an mRNA vaccine immunization group and a negative control group, and the negative control group was a PBS immunization group.

[0180] All mice in the mRNA vaccine immunization groups MPXV1 and MPXV2 were immunized with a dose of the mRNA vaccine prepared in Example 3 on the 0th day and the 14th day, respectively, by intramuscular injection on both sides of the thighs. The mice in the negative control group were injected with the same volume of PBS at the same time. The vaccination method was intramuscular injection, and the vaccination dose was 10 μg of mRNA vaccine or PBS per mouse each time. Mouse serum samples were collected on the 14th day and the 28th day, respectively, to test the binding antibody titer and neutralizing antibody titer of the immune serum. Blood sampling was performed 2 days before immunization. The number of immunizations was twice, and the immunization time and detection procedure time were as follows. Figure 3 The flow chart shown in Figure A.

[0181] (1) Specific IgG antibody reaction detection Antigens A35R, M1R, A29, and B6R were diluted to 10 μg / ml with ELISA coating buffer and added to 96-well plates and coated overnight at 4°C. After incubation with blocking buffer for 1 hour at room temperature, any uncoated proteins were washed away with washing buffer. Serum was diluted to four concentrations and added to the wells, and the corresponding antibodies were added at the same time. After incubation, the serum was washed out and HRP-goat anti-mouse IgG was added for final optical density analysis in an ELISA reader.

[0182] The results are as follows Figure 3 As shown in Figure B, the serum of MPXV1 mice collected on day 28 was diluted 1:2.57, 1:10000, 1:50000 and 1:200000. When the dilution factor was 1:50000, the OD of anti-A35R 450 The value is still greater than 0.5; when the dilution factor is 1:10000, the OD of anti-M1R 450 The value is about 1.5; when the dilution factor is 1:10000, the OD of anti-B6R 450 The value is still greater than 1.5, indicating that the MPXV1 vaccine of the present invention can induce the production of high-titer antigen-specific antibodies after immunization, has good immunogenicity, and can stimulate the body to produce specific antibodies against A35R, M1R, A29, and B6R antigens.

[0183] The corresponding formula was obtained based on the OD450 of the known antibodies and the corresponding antibody concentrations, and the corresponding concentrations of mouse serum were calculated. The results showed that according to the back-calculated concentrations, both the primary and secondary immunizations were significantly different from those before immunization. The serum antibodies after the secondary immunization were significantly increased compared with the primary immunization. The antibody levels corresponding to A35R, M1R, A29L, and B6R of MPXV1 and MPXV2 could reach 10 3 ng / ml, 10 4 ng / ml, 10 5 -107 ng / ml, 10 4 ng / ml, can reach 10 after the second immunization 4 ng / ml, 10 5 ng / ml, 10 7 -10 8 ng / ml, 10 5 ng / ml. And the difference within the experimental groups was small.

[0184] Results Figure 4 The results showed that both MPXV1 and MPXV2 vaccines could induce high levels of specific binding antibodies against each antigen (A35R, M1R, A29L, B6R) in mice, and the antibody levels against the four antigens were significantly improved after the second immunization.

[0185] (2) Neutralizing antibody titer detection In order to detect the neutralizing antibody content in the vaccine-induced serum antibodies, the mouse serum was tested 14 days after the second immunization, and the test was commissioned to Zhongshan Yiweidi Technology Co., Ltd. The specific operation was to inactivate the serum at 56°C for 30 minutes to remove complement and other potential neutralizing agents. A 4-fold gradient dilution was performed starting from 1:20 with serum-free medium, for a total of 6 gradients. Then 120μl of serum was mixed with an equal volume of poxvirus and incubated at 37°C for 1h. The mixture after incubation was added to the Vero 96-well cell plate (2×10 4 cells / mL), cultured at 37℃ for 72h, stained by immunoblotting, read the plate and record the number of fluorescent spots in each well, and calculate the antibody titer of the serum based on the ratio of the number of fluorescent spots in the wells containing different concentrations of serum to be tested to the number of fluorescent spots in the positive control wells without serum. 50 It refers to the antibody concentration that can cause half the maximal effect (i.e. 50% of the maximum inhibitory effect).

[0186] Results Figure 5 The results showed that the cells in the virus-free negative wells were normal and had no obvious lesions. The cells in the PBS group showed obvious cytopathic effects. 50 The value was 73, with no significant neutralizing activity. Both monkeypox MPXV1 and MPXV2 vaccines induced significant neutralizing antibodies after immunization, and the neutralizing antibody titer after the second immunization was greater than 2000. 50 The values ​​were 3136 and 2065 respectively. The neutralizing antibody titer of monkeypox MPXV1 after immunization was greater than that of MPXV2.

[0187] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A monkeypox virus monovalent multi-antigen fusion protein, characterized in that: The fusion protein has a structure as shown in Formula Ia or Formula Ib from N-terminus to C-terminus: R1-L1-R2 (Formula Ia) R2-L1-R1 (Formula Ib) in, R1 is the first fusion protein element comprising A35R and M1R proteins from N-terminus to C-terminus; L1 is a key or joint element; R2 is a second fusion protein element comprising A29L and B6R proteins from N-terminus to C-terminus; "-" represents a peptide bond.

2. The fusion protein according to claim 1, characterized in that The first fusion protein element comprises, from N-terminus to C-terminus: P1-A35R-linker1-M1R; Wherein, the P1 is a signal peptide, The A35R is the monkeypox virus A35R protein or its antigenic fragment or its derivative peptide segment, The linker1 is a flexible peptide linker. The M1R is the monkeypox virus M1R protein or its antigenic fragment or its derived peptide segment.

3. The fusion protein according to claim 2, characterized in that The amino acid sequence of A35R comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 16, or the amino acid sequence from positions 98 to 181 as shown in SEQ ID NO: 16; The amino acid sequence of M1R comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:17, or the amino acid sequence from position 1 to position 250 as shown in SEQ ID NO:

17.

4. The fusion protein according to claim 2, characterized in that The flexible peptide linker comprises 0-30 amino acids, preferably 1-10 amino acids.

5. The fusion protein according to claim 1, characterized in that The second fusion protein element comprises, from N-terminus to C-terminus: P2-A29L-linker2-B6R; Wherein, the P2 is a signal peptide, The A29L is the monkeypox virus A29L protein or its antigenic fragment or its derivative peptide segment, The linker2 is a flexible peptide linker. The B6R is the monkeypox virus B6R protein or its antigenic fragment or its derived peptide segment.

6. The fusion protein according to claim 5, characterized in that The amino acid sequence of A29L comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18, or the amino acid sequence from positions 1 to 40 as set forth in SEQ ID NO: 18; The amino acid sequence of B6R comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:19, or the amino acid sequence from positions 20 to 317 as shown in SEQ ID NO:

19.

7. The fusion protein according to claim 1, characterized in that The fusion protein comprises from N-terminus to C-terminus: P1-A35R-linker1-M1R-L1-P2-A29L-linker2-B6R; Wherein, P1 and P2 are each independently a signal peptide, The amino acid sequence of A35R is the amino acid sequence from position 98 to position 181 as shown in SEQ ID NO: 16, The amino acid sequence of the M1R is the amino acid sequence from position 1 to position 250 as shown in SEQ ID NO: 17, The amino acid sequence of A29L is the amino acid sequence from position 1 to position 40 as shown in SEQ ID NO: 18, The amino acid sequence of B6R is the amino acid sequence from position 20 to position 317 as shown in SEQ ID NO: 19, The linker1 and linker2 are each independently a flexible peptide linker, The L1 is a connector element.

8. An isolated nucleotide, characterized in that The nucleotides encode the fusion protein of claim 1; The nucleotide has the structure of Formula II: Z1-Z2-Z3-Z4-Z5 (II) In the formula, Z1 is none or 5' capping element; Z2 is the 5′-UTR element; Z3 is a nucleotide sequence encoding the fusion protein of claim 1; Z4 is the 3′-UTR element; Z5 is the polyA tail element; "-" means nothing or a concatenated sequence.

9. A pharmaceutical composition, characterized in that include: (a) the fusion protein of claim 1, or the nucleotide of claim 8; and (b) a pharmaceutically acceptable carrier.

10. A method for preparing the fusion protein according to claim 1, characterized in that: The following steps are involved: (a) culturing cells containing the nucleotide according to claim 8 integrated into their genome under appropriate conditions, thereby obtaining a culture containing the fusion protein; and (b) purifying and / or separating the culture obtained in step (a) to obtain the fusion protein targeting A35, M1, A29 and B6 monkeypox virus antigens.

11. A method for generating an immune response against monkeypox virus, characterized in that: Includes steps: The fusion protein of claim 1 or the nucleotide of claim 8 or the pharmaceutical composition of claim 9 is administered to a subject in need thereof.

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