A monovalent vaccine with multiple antigens against monkeypox virus and its application

Abstract

The present invention provides a monovalent multi-antigen vaccine against monkeypox virus and its application. Specifically, the present invention provides a monovalent multi-antigen fusion protein against monkeypox virus, and the fusion protein has a first fusion protein element containing A35R and M1R proteins and a second fusion protein element containing A29L and B6R proteins from the N-terminus to the C-terminus. In the present invention, A35R, M1R, A29L, and B6R are rationally combined as antigen components into a single protein molecule to prepare a monovalent mRNA vaccine. 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 high level of neutralizing antibodies.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically relates to a monovalent vaccine with multiple antigens of monkeypox virus and its application. Background Art

[0002] Monkeypox is a disease caused by the infection of monkeypox virus (MPXV). Monkeypox usually presents as a self-limiting infection, and the symptoms include fever, headache, fatigue, lymphadenopathy, and skin lesions. Monkeypox was first discovered in 1958 when two pox-like diseases broke out in monkey colonies raised for research, hence the name "monkeypox". Currently, all monkeypox vaccines approved (for emergency use) globally are live virus vaccines developed against smallpox virus, which have definite side effects and limited vaccinated populations. There are two main virus forms of monkeypox virus antigens: enveloped virus (EV) and mature virus (MV). The A35R protein from EV plays a certain role in the structure and function of the virus. Similarly, 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 process of virus adsorption and entry into host cells. Therefore, A29, M1, B6, and A35 are selected as key antigens for the design of monkeypox vaccines.

[0003] Currently, there is no monkeypox mRNA vaccine on the market. The preparation of conventional multi-antigen / multivalent mRNA vaccines is to separately perform steps such as template linearization, in vitro transcription, purification, and mixing and encapsulation for each antigen to prepare multi-antigen / multivalent mRNA vaccines. Such complex steps mean that with the increase in antigens, the production input will increase, the use of equipment will increase, and the production efficiency will decrease.

[0004] Therefore, there is an urgent need in this field to develop an MPXV multi-immunogen chimeric antigen and the corresponding mRNA vaccine with a simple preparation process, high production capacity, low cost, and capable of efficiently stimulating specific immune responses against monkeypox virus. Summary of the Invention

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

[0006] In the first aspect of the present invention, there is provided a monovalent multi-antigen fusion protein of monkeypox virus, and the fusion protein has a structure shown in Formula Ia or Formula Ib from the N-terminus to the C-terminus:

[0007] R1-L1-R2 (Formula Ia)

[0008] R2-L1-R1 (Formula Ib)

[0009] Wherein,

[0010] R1 is a first fusion protein element containing A35R and M1R proteins from the N-terminus to the C-terminus;

[0011] L1 is a bond or linker element;

[0012] R2 is a second fusion protein element containing A29L and B6R proteins from the N-terminus to the C-terminus;

[0013] "-" represents a peptide bond.

[0014] In another preferred embodiment, the monkeypox virus includes West African clade I, West African clade IIa, and West African clade IIb.

[0015] In another preferred embodiment, the monkeypox virus is West African clade IIb.

[0016] In another preferred embodiment, the monkeypox virus includes A.1, B.1, A1.1, A.2, B1.10, B1.7, B1.2, B1.3.

[0017] In another preferred embodiment, the monkeypox virus includes MPXV_USA_2022_MA001.

[0018] In another preferred embodiment, the first fusion protein element contains, from the N-terminus to the C-terminus: P1-A35R-linker1-M1R;

[0019] Wherein, P1 is a signal peptide,

[0020] The A35R is the A35R protein of monkeypox virus or its antigenic fragment (or its derivative peptide),

[0021] The linker1 is a flexible peptide linker,

[0022] The M1R is the M1R protein of monkeypox virus or its antigenic fragment (or its derivative peptide).

[0023] In another preferred embodiment, the signal peptide includes an amino acid sequence shown in SEQ ID NO: 1 or 2.

[0024] In another preferred example, the amino acid sequence of P1 is as shown in SEQ ID NO:1.

[0025] In another preferred example, 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 with the amino acid sequence of SEQ ID NO:16, or the amino acid sequence from position 98 to position 181 as shown in SEQ ID NO:16.

[0026] 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.

[0027] In another preferred example, the amino acid sequence of M1R comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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.

[0028] In another preferred example, the amino acid sequence of M1R is the amino acid sequence from position 1 to position 250 as shown in SEQ ID NO:17.

[0029] In another preferred example, the flexible peptide linker comprises 0 - 30 amino acids, preferably 1 - 10 amino acids.

[0030] In another preferred example, the flexible peptide linker is 1 - 4 GGGGS and / or GGGS.

[0031] In another preferred example, the linker1 is (G3S)4, and the sequence is as shown in SEQ ID NO:3.

[0032] In another preferred example, the second fusion protein element comprises, from the N - terminus to the C - terminus:

[0033] P2 - A29L - linker2 - B6R;

[0034] wherein, P2 is a signal peptide,

[0035] A29L is the monkeypox virus A29L protein or its antigenic fragment (or its derivative peptide),

[0036] linker2 is a flexible peptide linker,

[0037] B6R is the monkeypox virus B6R protein or its antigenic fragment (or its derivative peptide).

[0038] In another preferred example, the amino acid sequence of P2 is as shown in SEQ ID NO:2.

[0039] In another preferred example, the amino acid sequence of A29L comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:18, or the amino acid sequence from position 1 to position 40 as shown in SEQ ID NO:18.

[0040] 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.

[0041] In another preferred example, the amino acid sequence of B6R comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:19, or the amino acid sequence from position 20 to position 317 as shown in SEQ ID NO:19.

[0042] 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.

[0043] In another preferred example, the linker2 is (G3S)4, and the sequence is as shown in SEQ ID NO:3.

[0044] In another preferred example, the fusion protein comprises, from the N-terminus to the C-terminus:

[0045] P1 - A35R - linker1 - M1R - L1 - P2 - A29L - linker2 - B6R;

[0046] wherein, the P1 and P2 are each independently a signal peptide,

[0047] the amino acid sequence of A35R is the amino acid sequence from position 98 to position 181 as shown in SEQ ID NO:16,

[0048] the amino acid sequence of M1R is the amino acid sequence from position 1 to position 250 as shown in SEQ ID NO:17,

[0049] the amino acid sequence of A29L is the amino acid sequence from position 1 to position 40 as shown in SEQ ID NO:18,

[0050] The amino acid sequence of the B6R is the amino acid sequence from the 20th to the 317th amino acid shown in SEQ ID NO:19,

[0051] The linker1 and linker2 are each independently a flexible peptide linker,

[0052] The L1 is a linker element.

[0053] In another preferred example, the signal peptide includes the amino acid sequence shown in SEQ ID NO:1 or 2.

[0054] In another preferred example, the amino acid sequence of the P1 is as shown in SEQ ID NO:1.

[0055] In another preferred example, the amino acid sequence of the P2 is as shown in SEQ ID NO:2.

[0056] In another preferred example, the fusion protein has the activity of simultaneously binding to A35, M1, A29 and B6 monkeypox virus antigens.

[0057] In another preferred example, the flexible peptide linker includes 0 - 30 amino acids, preferably 1 - 10 amino acids.

[0058] In another preferred example, the flexible peptide linker is 1 - 4 GGGGS and / or GGGS.

[0059] In another preferred example, the linker1 is (G3S)4, and the sequence is as shown in SEQ ID NO:3.

[0060] In another preferred example, the linker2 is (G3S)4, and the sequence is as shown in SEQ ID NO:3.

[0061] In another preferred example, the linker element is a 2A polypeptide or its derivative polypeptide.

[0062] In another preferred example, the amino acid sequence of the linker element is as shown in SEQ ID NO:4.

[0063] In another preferred example, the fusion protein from the N-terminus to the C-terminus contains: P1 - A35R - (G3S)4M1R - 2A - P2 - A29L - (G3S)4 - B6R,

[0064] wherein, the A35R protein and the M1R protein are connected by (G3S)4 to form a first fusion protein element,

[0065] the A29L protein and the B6R protein are connected by (G3S)4 to form a second fusion protein element,

[0066] The first fusion protein and the second fusion protein are linked by a linker element 2A.

[0067] In another preferred embodiment, the amino acid sequence of P1 is as shown in SEQ ID NO:1.

[0068] In another preferred embodiment, the amino acid sequence of P2 is as shown in SEQ ID NO:2.

[0069] In another preferred embodiment, the amino acid sequence of the fusion protein is as shown in SEQ ID NO:5.

[0070] In a second aspect of the present invention, there is provided an isolated nucleotide encoding the fusion protein described in the first aspect of the present invention.

[0071] In another preferred embodiment, the nucleotide has a structure of formula II:

[0072] Z1-Z2-Z3-Z4-Z5 (II)

[0073] In the formula,

[0074] Z1 is none or a 5'-capping element;

[0075] Z2 is a 5'-UTR element;

[0076] Z3 is a nucleotide sequence encoding the fusion protein described in the first aspect of the present invention;

[0077] Z4 is a 3'-UTR element;

[0078] Z5 is a polyA tail element;

[0079] "-" is none or a linking sequence.

[0080] 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.

[0081] In another preferred embodiment, the nucleotide is mRNA.

[0082] In another preferred embodiment, the nucleotide sequence is as shown in SEQ ID NO:7.

[0083] In another preferred embodiment, the mRNA is encapsulated in liposomes to form liposome nanoparticles.

[0084] In another preferred embodiment, the average particle size of the liposome nanoparticles is 10 - 500 nm, preferably 60 - 100 nm, more preferably 60 - 80 nm.

[0085] In another preferred embodiment, the liposome nanoparticles comprise an outer liposome layer and the mRNA located inside.

[0086] In another preferred embodiment, the components of the liposome layer include any liposome layer, preferably selected from the group consisting of PEG-modified lipids (such as DMG-PEG 2000), neutral lipids (such as DSPC), cationic lipids (such as SM102), cholesterol, or combinations thereof.

[0087] In another preferred embodiment, the liposome layer of the nanoparticles includes any liposome layer, preferably including cationic lipids, cholesterol, phosphatidylcholine, and PEG-modified lipids.

[0088] In another preferred embodiment, the PEG-modified lipids are selected from the group consisting of DMG-PEG 2000, C14-PEG2000, C16-PEG2000, or combinations thereof.

[0089] In another preferred embodiment, the PEG-modified lipid is DMG-PEG 2000.

[0090] In another preferred embodiment, the cationic lipids are selected from the group consisting of SM102, DODAP, D-Lin-MC3-DMA, DODMA, or combinations thereof.

[0091] In another preferred embodiment, the cationic lipid is SM102.

[0092] In the third aspect of the present invention, there is provided a vector containing the nucleotide as described in the second aspect of the present invention.

[0093] In another preferred embodiment, the vector is: pET vector, pGEM-T vector, pcDNA3.1, or combinations thereof.

[0094] In the fourth aspect of the present invention, there is provided a host cell containing 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 integrated into the genome.

[0095] In the fifth aspect of the present invention, there is provided a pharmaceutical composition comprising the following components:

[0096] (a) 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; and

[0097] (b) A pharmaceutically acceptable carrier.

[0098] In another preferred embodiment, the content of component (a) is 0.1-99.9 wt%, preferably 10-99.9 wt%, more preferably 70%-99.9 wt%.

[0099] In another preferred embodiment, the dosage form of the pharmaceutical composition is liquid, solid, or gel state.

[0100] In another preferred embodiment, the pharmaceutical composition is an mRNA vaccine composition.

[0101] In another preferred embodiment, the vaccine composition comprises the nucleotide (mRNA) as described in the second aspect of the present invention and liposomes.

[0102] In another preferred embodiment, the vaccine composition is liposome nanoparticles.

[0103] In another preferred embodiment, the average particle size of the liposome nanoparticles is 10-500 nm, preferably 60-100 nm, more preferably 60-80 nm.

[0104] In another preferred embodiment, the liposome nanoparticles include an outer liposome layer and the mRNA located inside.

[0105] In another preferred embodiment, the components of the liposome layer include any liposome layer, preferably selected from the group consisting of PEG-modified lipids (such as DMG-PEG 2000), neutral lipids (such as DSPC), cationic lipids (such as SM102), cholesterol, or combinations thereof.

[0106] In another preferred embodiment, the liposome layer of the nanoparticles includes any liposome layer, preferably including cationic lipids, cholesterol, phosphatidylcholine, and PEG-modified lipids.

[0107] In another preferred embodiment, the PEG-modified lipids are selected from the group consisting of DMG-PEG 2000, C14-PEG2000, C16-PEG2000, or combinations thereof.

[0108] In another preferred embodiment, the PEG-modified lipid is DMG-PEG 2000.

[0109] In another preferred embodiment, the cationic lipids are selected from the group consisting of SM102, DODAP, D-Lin-MC3-DMA, DODMA, or combinations thereof.

[0110] In another preferred embodiment, the cationic lipid is SM102.

[0111] 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 nasal and oral spray and aerosol inhalation.

[0112] In another preferred embodiment, the mRNA itself in the vaccine composition can also act as an adjuvant.

[0113] In another preferred embodiment, the subject to which the pharmaceutical composition or formulation is administered includes humans or non-human animals.

[0114] In another preferred embodiment, the non-human animals include: rodents (such as rats, mice), primates (such as monkeys).

[0115] In another preferred embodiment, the pharmaceutical composition is used for the prevention and / or treatment of monkeypox virus infection.

[0116] In another preferred embodiment, the prevention and / or treatment of monkeypox virus infection includes a delay in the development of related symptoms following monkeypox virus infection and / or a reduction in the severity of these symptoms.

[0117] In another preferred embodiment, the prevention and / or treatment of monkeypox virus infection further includes alleviating the accompanying symptoms of existing monkeypox virus infection and preventing the appearance of other symptoms.

[0118] In another preferred embodiment, the pharmaceutical composition or formulation can be co-administered with other antiviral or anti-inflammatory drugs.

[0119] In another preferred embodiment, the other antiviral or anti-inflammatory drugs for co-administration are selected from the group consisting of: viral replication inhibitors, hormonal anti-inflammatory drugs, biological response modifiers, monoclonal antibodies, or combinations thereof.

[0120] In the sixth aspect of the present invention, a method for preparing the fusion protein described in the first aspect of the present invention is provided, comprising the following steps:

[0121] (a) Culturing the host cell described in the fourth aspect of the present invention under suitable conditions to obtain a culture containing the fusion protein; and

[0122] (b) Purifying and / or isolating the culture obtained in step (a) to obtain the fusion protein targeting A35, M1, A29, and B6 monkeypox virus antigens.

[0123] In the seventh aspect of the present invention, there is provided the use of a fusion protein as described in the first aspect of the present invention, or a nucleotide as described in the second aspect of the present invention, or a pharmaceutical composition as described in the fifth aspect of the present invention, for preparing a medicament, a reagent, a test plate or a kit; wherein, the reagent, the test 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.

[0124] In another preferred example, the medicament is used to block the invasion of monkeypox virus into the human body.

[0125] In another preferred example, the reagent is one or more reagents selected from the group consisting of: an isotope tracer, a contrast agent, a flow cytometry detection reagent, a cell immunofluorescence detection reagent, a magnetic nanoparticle and an imaging agent.

[0126] In another preferred example, the reagent for detecting monkeypox virus in the sample is a contrast agent for detecting monkeypox virus (in vivo).

[0127] In another preferred example, the detection is in vivo detection or in vitro detection.

[0128] In another preferred example, the detection includes flow cytometry detection, cell immunofluorescence detection, or a combination thereof.

[0129] In another preferred example, the monkeypox virus or its related diseases are selected from the group consisting of: monkeypox virus infection, other poxvirus (such as vaccinia virus) infection, or a combination thereof.

[0130] In another preferred example, the monkeypox virus includes West African clade I, West African clade IIa, and West African clade IIb.

[0131] In another preferred example, the monkeypox virus includes A.1, B.1, A1.1, A.2, B1.10, B1.7, B1.2, B1.3.

[0132] In the eighth aspect of the present invention, there is provided a method for generating an immune response against monkeypox virus, comprising the step of administering to a subject in need 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.

[0133] In another preferred example, the method includes administering to a subject in need liposome nanoparticles containing the mRNA.

[0134] In another preferred example, the subject includes a human or a non-human mammal.

[0135] In another preferred example, the non-human mammal includes a non-human primate (such as a monkey).

[0136] In another preferred example, the method induces neutralizing antibodies against monkeypox virus in the subject.

[0137] In a ninth aspect of the present invention, there is provided a method for preventing and / or treating monkeypox virus infection, comprising the step of administering to a subject in need thereof 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.

[0138] In another preferred example, the method comprises administering to a subject in need thereof liposome nanoparticles containing the mRNA.

[0139] In another preferred example, the prevention and / or treatment of monkeypox virus infection includes blocking the invasion of monkeypox virus into the human body.

[0140] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] Figure 1Shows the schematic diagram of the candidate mRNA sequence structure and the optimization result diagram in the embodiments of the present invention. Among them, A is the result diagram of detecting the influence of two proteins formed by connecting A35R and M1R with 2A or connecting antigen protein B6R and M1R with 2A on the expression level of M1R. The results show that the expression level of M1R corresponding to A35R in front of M1R is higher than that corresponding to B6R in front of M1R; B is the result diagram of comparing the expression levels of B6R corresponding to 3 signal peptides. Among them, B6R sig is the self-signal peptide of B6R, mouse immunoglobulin heavy chain sig is the signal peptide of mouse immunoglobulin heavy chain, and tPA sig is the signal peptide of human tissue plasminogen activator. The results show that the expression level of B6R with mouse immunoglobulin heavy chain sig is the highest. Therefore, this signal peptide is selected as the signal peptide of A29L-(G3S)4-B6R fusion protein; C and D respectively show the result diagrams of comparing the expression levels of A35R and M1R by selecting 2 signal peptides in the screening of A35R(98-181aa)-linker-M1R. Among them, mouse immunoglobulin heavy chain sig is the signal peptide of mouse immunoglobulin heavy chain, and mIL-21R sig is the signal peptide of mouse interleukin 21 receptor. The results show that the expression level of A35R(98-181aa)-linker-M1R with mIL-21R sig is the highest. Therefore, this signal peptide is selected as the signal peptide of A35R(98-181aa)-linker-M1R fusion protein. A35R(98-181aa) represents that the A35 sequence is the amino acid sequence from the 98th to the 181st position relative to the sequence shown in SEQ ID NO:16; E is the schematic diagram of the candidate mRNA sequence structure. 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.

[0142] Figure 2 Shows the Western blot detection results of the proteins expressed by the monkeypox vaccine antigen mRNA at the in vitro cell level. 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.

[0143] Figure 3 Shows the flow chart of mouse immunization and blood collection and the result diagram of detecting the humoral immune response induced by the mRNA vaccine in mice. Among them, A is the flow chart of mouse immunization and blood collection, and B is the result diagram of detecting the antibody titers after collecting serum and diluting it by different multiples on the 28th day of immunization.

[0144] Figure 4Shows the results of the humoral immune response in mice induced by the mRNA vaccine measured by enzyme-linked immunosorbent assay (ELISA). "*" indicates comparison with the control group PBS in the same time period. The horizontal line indicates pairwise comparison. "*" indicates P < 0.05; "**" indicates P < 0.01, and "***" indicates P < 0.001.

[0145] Figure 5 Shows the results of the monkeypox virus neutralizing antibody assay. "*" indicates P < 0.05 compared with the control group PBS. Detailed implementation mode

[0146] Through a large number of experimental screenings, the present invention first selects 4 proteins (A35R, M1R, A29L, and B6R) with strong immunogenicity in the surface protein of the monkeypox virus as candidate target proteins, modifies the proteins, and screens the arrangement order and signal peptides. Finally, a monkeypox virus mRNA vaccine is obtained by sequentially concatenating the 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 host cells and enhance its immunogenicity. The experimental results show that the mRNA vaccine of the present invention can ensure the high-efficiency 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.

[0147] Terms

[0148] 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 the monkeypox virus monovalent antigen fusion protein having the structure of Formula Ia or Formula Ib shown in the first aspect of the present invention.

[0149] As used herein, the terms "nucleotide of the present invention", "nucleotide encoding the fusion protein", "mRNA of the present invention", "mRNA for preventing monkeypox virus of the present invention", "mRNA molecule of the present invention", and "mRNA sequence of the present invention" are used interchangeably and all refer to the nucleotide encoding the fusion protein described in the first aspect of the present invention.

[0150] Monkeypox virus

[0151] Monkeypox virus is a zoonotic virus belonging to the genus Orthopoxvirus, a double-stranded DNA virus, similar to variola virus (VARV), cowpox virus (CPX), and vaccinia virus (VACV). MPV is oval-shaped with an outer lipoprotein membrane. The genome is approximately 190 kb. Monkeypox can be divided into two distinct clades: Central Africa (also known as the Congo Basin) and West Africa, with a high degree of sequence similarity between the two clades. The West African clade has caused the current global spread of the epidemic.

[0152] The clinical manifestations of monkeypox virus infection 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 initially named monkeypox virus after being isolated from monkeys, and the main carriers of the virus are rodents.

[0153] The monovalent antigen fusion protein of monkeypox virus of the present invention

[0154] In the present invention, a class of fusion proteins or their conjugates that simultaneously bind to A35, M1, A29, and B6 monkeypox virus antigens is provided.

[0155] Preferably, the fusion protein comprises, from the N-terminus to the C-terminus: P1 - A35R - linker1 - M1R - L1 - P2 - A29L - linker2 - B6R;

[0156] Wherein, the P1 and P2 are each independently a signal peptide,

[0157] The amino acid sequence of A35R is the amino acid sequence from position 98 to position 181 as shown in SEQ ID NO:16,

[0158] The amino acid sequence of M1R is the amino acid sequence from position 1 to position 250 as shown in SEQ ID NO:17,

[0159] The amino acid sequence of A29L is the amino acid sequence from position 1 to position 40 as shown in SEQ ID NO:18,

[0160] The amino acid sequence of B6R is the amino acid sequence from position 20 to position 317 as shown in SEQ ID NO:19,

[0161] The linker1 and linker2 are each independently a flexible peptide linker,

[0162] The L1 is a linker element.

[0163] Preferably, the signal peptide comprises the amino acid sequence as shown in SEQ ID NO:1 or 2.

[0164] Preferably, the amino acid sequence of P1 is as shown in SEQ ID NO:1.

[0165] Preferably, the amino acid sequence of P2 is as shown in SEQ ID NO:2.

[0166] Preferably, the fusion protein of the present invention has the amino acid sequence as shown in SEQ ID NO:5.

[0167] As used herein, the term "fusion protein" also includes variant forms having antigen-binding activity against A35, M1, A29, and B6 monkeypox viruses. These variant forms include (but are not limited to): deletion, insertion, and / or substitution of 1-3 (usually 1-2, more preferably 1) amino acids, addition or deletion of one or several (usually within 3, preferably within 2, more preferably within 1) amino acids at the C-terminus and / or N-terminus, or addition of an amino acid fragment with a relatively small amino acid side chain as a linker (such as glycine, serine, etc.) at the N-terminus or C-terminus of the small protein. For example, in the art, when substituting amino acids with similar or comparable properties, the function of the protein is usually not changed. Also, for example, addition or deletion of 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 monomeric and polymeric forms of the polypeptides of the present invention. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0168] The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are replaced, or (ii) polypeptides having substituent groups in one or more amino acid residues, or (iii) polypeptides formed by fusing the 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 fusing an additional amino acid sequence to this polypeptide sequence (a fusion protein formed by fusing with a leader sequence, a secretion sequence, or a tag sequence such as 6His). According to the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0169] A preferred class of active derivatives refers to polypeptides in which, compared with the amino acid sequence of the present invention, up to 5, preferably up to 3, more preferably up to 1 amino acid is replaced by an amino acid with similar or comparable properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0170] Table 1

[0171]

[0172] The present invention also provides analogs of the fusion protein of the present invention. These analogs may differ from the polypeptides of the present invention in terms of amino acid sequence, or in terms of modified forms that do not affect the sequence, or both. The analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0173] The term "polynucleotide of the present invention" may be a polynucleotide encoding the fusion protein of the present invention, or may also be a polynucleotide further including additional coding and / or non-coding sequences.

[0174] The present invention also relates to variants of the above 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 is known in the art, allelic variants are alternative forms of a polynucleotide, which may be substitutions, deletions or insertions of one or more nucleotides, but do not substantially change the function of the ultra-high-affinity small protein or fusion protein encoding the targeted S protein.

[0175] The present invention also relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, 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 high-stringency conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and washing at low ionic strength and high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of a denaturing agent 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% or more, preferably 95% or more.

[0176] mRNA vaccine

[0177] 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.

[0178] Typically, the method of the present invention includes: first obtaining the expression gene (or coding sequence) of the antigen protein by PCR method, then obtaining the corresponding mRNA by in vitro transcription technology, and finally obtaining an efficient mRNA vaccine.

[0179] mRNA vaccines are mRNA with expression activity prepared in vitro, and its main structure includes 5' and 3' UTRs as well as 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 activate both MHC I and MHC II presentation pathways simultaneously, thus greatly improving the immunogenicity of the vaccine.

[0180] In order to increase the expression level of the mRNA vaccine, in the present invention, a series of nucleic acid elements that promote mRNA expression are selected. The elements include (but are not limited to): 5'UTR, signal peptide, antigen sequence, and 3'UTR.

[0181] In the present invention, the preferred proteins of the monkeypox virus are the proteins in the order of A35R, M1R, A29L, and B6R. The mRNA vaccine in the present invention includes mRNA encoding these proteins of the monkeypox virus in tandem.

[0182] In the present invention, SEQ ID NO: 12 - 15 are the mRNA sequences of the A35R, M1R, A29L, and B6R proteins of the monkeypox virus respectively.

[0183] In a preferred example, the mRNA vaccine of the present invention includes the mRNA of the A35R, M1R, A29L, and B6R proteins of the monkeypox virus.

[0184] In the present invention, the monkeypox virus includes West African clade I, West African clade IIa, and West African clade IIb.

[0185] 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.

[0186] In a preferred embodiment, the monkeypox virus includes MPXV_USA_2022_MA001.

[0187] In a preferred embodiment, the present invention selects the monkeypox virus strain (MPXV_USA_2022_MA001) on NCBI as the reference sequence because this sequence best conforms to the current reality of the monkeypox epidemic and is expected to play a greater role in controlling the epidemic.

[0188] In the present invention, four monkeypox virus proteins were selected as target proteins. Using the monkeypox virus strain (MPXV_USA_2022_MA001 (Genbank NO: 563414.2)) from the 2022 epidemic strain as the reference genome, their protein names were named A35R, M1R, A29L, and B6R respectively.

[0189] The above protein sequences were modified and designed based on the original monkeypox virus sequences to overcome many defects and achieve the maximum effect of vaccine-induced immune responses. The modified nucleic acid sequences are shown in SEQ ID NO: 6.

[0190] In addition, the present invention compared multiple vaccine regimens, including four monovalent regimens, two bivalent regimens, and one quadrivalent regimen.

[0191] In the monovalent regimens, vaccines were designed with each protein (A35R, M1R, A29L, B6R) alone as the target protein.

[0192] In the bivalent regimens, two proteins were combined: A35R and M1R; or A29L and B6R.

[0193] In the quadrivalent regimen, four proteins were combined: A35R, M1R, A29L, B6R.

[0194] The vaccine design scheme of the present invention is different from the existing techniques in the combination mode of protein vaccines. The present invention belongs to a monovalent multi-antigen vaccine.

[0195] In a preferred example, the amino acid sequence of the monkeypox virus vaccine of the present invention is shown in SEQ ID NO: 5.

[0196] In addition, the present invention was modified by the following method steps to overcome the problem of low expression efficiency of the target protein in host cells and enhance its immunogenicity. The steps are as follows:

[0197] 1) Introduce a signal peptide into the expression plasmid;

[0198] 2) Remove the transmembrane region;

[0199] 3) Introduce polyA into the expression plasmid;

[0200] To improve the expression efficiency and expression level of the target protein in host cells and induce a stronger immune response, for the A35R and M1R fusion proteins, a signal peptide sequence (SEQ ID NO: 1) was added to their N-terminus; for the A29L and B6R fusion proteins, a signal peptide sequence (SEQ ID NO: 2) was added to their N-terminus to enhance their immunogenicity.

[0201] The present invention also includes related products produced using the designed monovalent multi-antigen vaccine, including purified antigen proteins, polyclonal and monoclonal antibody reagents against monkeypox antigens, and related drugs.

[0202] The vaccine of the present invention induces the production of specific neutralizing antibodies with high affinity for the immunogen in mice. The present invention also includes related specific antigens, a preparation method for the induced neutralizing antibodies, and a design scheme based on the variable region gene sequences of these high-affinity neutralizing antibodies.

[0203] Compositions and administration methods

[0204] The present invention also provides a composition, which comprises the liposome nanoparticles of the present invention and the mRNA vaccine of the present invention.

[0205] The compositions of the present invention include pharmaceutical compositions and vaccine compositions.

[0206] The compositions of the present invention may contain nucleotides encoding multiple antigen proteins (a combination of two or more of A35R, M1R, A29L, and B6R, or a combination of the four proteins A35R, M1R, A29L, and B6R).

[0207] The pharmaceutical compositions or vaccine compositions of the present invention can be prepared into various dosage forms, including (but not limited to): injections, suspensions, sprays, etc.

[0208] The main advantages of the present invention include:

[0209] (1) The present invention provides a multi-immunogen fusion antigen against monkeypox virus, which sequentially includes four immunogens: 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 unique to intracellular mature virus particles (MV), and M1R and A29L are antigens unique to extracellular enveloped virus particles (EV); the vaccine containing these four immunogens can stimulate an immune response against two infectious virus particles.

[0210] (2) The present invention forms a monovalent multi-antigen by directly connecting multiple monkeypox antigen proteins or their antigenic fragments in series or through an appropriate linker sequence. The obtained 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.

[0211] (3) 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, the present invention selects the proteins with strong immunogenicity in the surface proteins of the monkeypox virus as candidate target proteins to develop vaccines. The sequence is completely different from the reference sequences and modification methods used in the design of vaccine target protein sequences in the prior art. The present invention designs a monovalent multi-antigen vaccine, and through different combinations of target proteins, induces an immune response in the body to obtain high-titer antibodies that can block the infection of the monkeypox virus and enhance the immune effect of the vaccine.

[0212] (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 method with the optimal protection effect, and obtains high-titer antibodies that can block virus infection.

[0213] (5) The present invention designs and modifies the target protein, and by removing the transmembrane region of the target protein and introducing sequences such as signal peptides in the original sequence, improves the stability and efficiency of protein expression of the recombinant plasmid, and ensures that the target protein can be highly expressed in the body.

[0214] (6) The present invention uses one mRNA to encode multiple protective antigens of the monkeypox virus MPXV, which can produce a high level of immune protection effect against the monkeypox virus and can be used to prepare vaccines or drugs for preventing and treating monkeypox. The mRNA vaccine composition prepared by the present invention can be used for vaccination of the general population when needed, which is safer. Therefore, it has broad application prospects in clinical practice.

[0215] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out according to the conditions described in "Molecular Cloning: A Laboratory Manual" (translated by Huang Peitang et al., Beijing: Science Press, 2002) by Sambrook.J et al. in the United States, or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0216] The experimental materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.

[0217] The preparation method of the monkeypox monovalent vaccine provided by the present invention mainly includes amino acid sequences, DNA sequences, and mRNA sequences. The amino acid sequences mainly refer to the antigens selected in antigen design, preferably A35R (98-181aa, that is, the 98-181st positions of the amino acid sequence shown in SEQ ID NO:16), M1R (1-250aa, that is, the 1-250th positions of the amino acid sequence shown in SEQ ID NO:17), A29L (1-40aa, that is, the 1-40th positions of the amino acid sequence shown in SEQ ID NO:18), B6R (20-317aa, that is, the 20-317th positions of the amino acid sequence shown in SEQ ID NO:19). Four antigens are combined and linked. The linker is preferably (G3S)4 (the sequence is shown in SEQ ID NO:3) and 2A peptide sequence (the sequence is shown in SEQ ID NO:4). First, (G3S)4 is used to fuse two antigens (A35R and M1R, or A29L and B6R). There are 2 groups of fusions corresponding to the 4 antigens, namely the fusion protein A35R-(G3S)4-M1R and the fusion protein A29L-(G3S)4-B6R; then the 2A sequence is used to link the 2 groups of fusion antigens to obtain a protein amino acid chain MPXCV1. The preferred linking order of the present invention is A35R-(G3S)4-M1R-2A-A29L-(G3S)4-B6R.

[0218] Example 1 Preparation of Monkeypox Virus mRNA

[0219] (1)Design of Amino Acid Sequences of Candidate Antigen Fusion Proteins and Plasmid Construction

[0220] The amino acid sequences of the A35R, M1R, A29L, and B6R proteins selected in the present invention are derived from the monkeypox 2022 epidemic strain MPXV_USA_2022_MA001 (Genbank NO:563414.2). Among them, the full-length amino acid sequences of the A35R, M1R, A29L, and B6R proteins are shown in SEQ ID NOs:16-19 respectively.

[0221] First of all, the inventors optimized the order arrangement of the A35R, M1R, A29L, and B6R proteins, and used 2A to link A35R and M1R to form a fusion protein: A35R-2A-M1R; used 2A to link B6R and M1R to form a fusion protein: B6R-2A-M1R, and compared the expression levels of M1R in the two.

[0222] The results of Western Blot are as Figure 1As shown in Figure A, when Protein A35R is placed in front of M1R compared to B6R placed in front of M1R, placing A35R in front of M1R results in a higher expression level of M1R. Therefore, in the present invention, the amino acid sequence of A35R is placed at the first position, and through screening, the amino acid sequence at positions 98 - 181 of the A35R amino acid sequence shown in SEQ ID NO:16 (abbreviated as A35R(98 - 181)) is finally selected and placed at the first position of the candidate antigen fusion protein of the present invention.

[0223] Secondly, in the screening of B6R, 3 signal peptides were selected for comparison of expression levels, namely the signal peptide of B6R itself (B6R sig), the signal peptide of the 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 the mouse immunoglobulin heavy chain was the highest ( Figure 1 Figure B), so this signal peptide was selected as the signal peptide of the A29L-(G3S)4-B6R fusion protein.

[0224] In the screening of A35R(98 - 181)-(G3S)4-M1R, 2 signal peptides were selected for comparison of expression levels, namely the signal peptide of the immunoglobulin heavy chain from mice (mouse immunoglobulin heavy chain signal peptide) and the signal peptide of interleukin-21 receptor from mice (mIL-21R sig). Through Western blot experiments, the expression levels of A35R ( Figure 1 Figure C) and M1R ( Figure 1 Figure D) were detected. Whether it was A35R or M1R, the expression level was higher when using mIL-21R sig than when using the mouse immunoglobulin heavy chain signal peptide. Therefore, this signal peptide was selected as the signal peptide of the A35R(98 - 181)-(G3S)4-M1R fusion protein.

[0225] The present invention preferably arranges the amino acid sequence of the candidate antigen fusion protein from the N-terminus to the C-terminus in the order of A35R-M1R-A29L-B6R. Among them, the A35 signal peptide is preferably mIL-21R sig (the sequence is shown in SEQ ID NO:1), which is fused to the N-terminus. The A29L signal peptide is preferably the signal peptide of the mouse immunoglobulin heavy chain (the sequence is shown in SEQ ID NO:2). The linker peptide between A35R and M1R and the linker peptide between A29L and B6R are preferably (G3S)4 (the sequence is shown in SEQ ID NO:3). The linker peptide between A29L and B6R is preferably the 2A peptide sequence (the sequence is shown in SEQ ID NO:4).

[0226] The antigen fusion protein sequence is shown in SEQ ID NO:5. SEQ ID NO:5 was optimized with human codons to obtain a nucleic acid sequence (the sequence is shown in SEQ ID NO:6), and this nucleic acid sequence was constructed onto a vector. The upstream of SEQ ID NO:6 contains a 5'UTR sequence (including the Kozak sequence), and the downstream contains a stop codon TGATGA sequence, a 3'UTR sequence, a PolyA sequence, and a BsaI restriction site to obtain a candidate target mRNA sequence, named MPXV1 (the sequence is shown in SEQ ID NO:7), which was constructed onto the basic plasmid pUC57 (containing the T7 promoter) by conventional molecular biology 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 from the 2022 epidemic strain of monkeypox MPXV_USA_2022_MA001 (Genbank NO: 563414.2) were respectively constructed onto vectors, and the signal peptides of M1R and B6R were preferably the mouse immunoglobulin heavy chain signal peptide. The amino acid sequences of A35R, M1R, A29L, and B6R proteins with signal peptide modification are shown in SEQ ID NO:8 - 11 respectively, and the corresponding nucleic acid sequences are shown in SEQ ID NO:12 - 15 respectively. Subsequently, the samples of the 4 mRNAs of A35R, M1R, A29L, and B6R mixed and encapsulated were named MPXV2. The schematic diagram of the candidate mRNA sequence structure is shown in Figure 1 Figure E in

[0227] (2)Plasmid extraction

[0228] After the plasmid construction was completed and the sequence was confirmed to be correct by sequencing, the strain was preserved. The strain was inoculated into 2YT medium, and kanamycin was added to the medium. The inoculation was carried out at a ratio of 100 μL of bacterial solution per 200 ml of volume of the medium, and the culture was incubated overnight at 37 °C on a shaker for 14 - 16 hours until the OD600 was between 2.0 - 3.0, and the bacterial cells were collected by centrifugation. The bacterial cells were resuspended with P1 buffer (25 mM Tris-HCl (pH 8.0), 10 mM EDTA, 50 mM glucose), then lysed by adding P2 buffer (250 mM NaOH, 1% (w / v) SDS (sodium dodecyl sulfate)), and finally neutralized by adding P3 buffer (3M potassium acetate, 5M acetic acid). Then, the supernatant was taken by centrifugation for Bestarose 6FF gel filtration, and finally the sample was transferred to a 100KD ultrafiltration tube for concentration. After concentration, the sample was aspirated into a new collection tube, and the concentration was measured and stored.

[0229] (3)Preparation of linearized plasmid template

[0230] The supercoiled plasmid was digested with enzymes. The 50 μL plasmid linearization system is shown in Table 2 below:

[0231] Table 2

[0232]

[0233] Add each component to a centrifuge tube. After mixing, incubate at 37 ± 0.5 °C in an incubator for 16 h. After the reaction, take a sample for agarose gel electrophoresis to confirm the linearization effect. Among them, the electrophoresis result of MPXV1 shows a single band with the correct size. Use isopropanol precipitation to recover the completely linearized plasmid, and detect the residual RNase in the recovered linearized plasmid. The linearized plasmid without obvious degradation is used for the next step

[0234] (4)In vitro transcription

[0235] The co-transcription capping in vitro transcription system is shown in Table 3 below:

[0236] Table 3

[0237]

[0238] After mixing the co-transcription capping in vitro transcription system, place it in an incubator and let it stand at 37 °C ± 0.5 °C for 3 h. After the transcription reaction, use DNase I to digest the template. After the transcription ends, add DNase I, GMP grade (2 U / μL), mix well, and react at 37 ± 0.5 °C for 30 min. Take out 1 μL of the sample, dilute it 10 times and load the sample for agarose gel electrophoresis to observe whether the IVT template digestion is complete. Then use an OligodT column to purify and remove impurities such as proteins and salts. Confirm that the purity of the prepared mRNA > 90% and there is no obvious degradation through gel electrophoresis and Qsep analysis

[0239] Example 2 mRNA in vitro cell transfection experiment

[0240] (1)mRNA transfection

[0241] 293T cells are cultured in a 75 cm 2 vented-cap right-angle culture flask with DMEM medium supplemented with 10% fetal bovine serum (FBS). When the cells grow to 70–90% confluence, digest the cells with trypsin to make a cell suspension with a cell suspension density of 2.5x10 5, Seed the cells in a 24-well plate at 500 μL per well and incubate overnight. The next day, discard the original medium before transfection and replace it with Opti-MEM™ I reduced-serum medium. Slowly add 450 μl of Opti-MEM™ I medium to the cells in each well along the sidewall. Prepare 2 tubes and dilute the transfection reagent and the mRNA sample with Opti-MEM™ I medium respectively. Add 1.5 μL of the transfection reagent Lipofectamin mRNA Max (Thermo Fisher Scientific) to 25 μL of Opti-MEM™ I medium, and add 2 μg of the mRNA stock solution prepared in Example 1 to 25 μL of Opti-MEM™ I medium, wherein A35R, M1R, A29L, and B6R of MPXV2 are mixed at a mass ratio of 1:1:1:1. Pipette and mix the mixtures in the two tubes respectively, and let stand for 10 min. After the waiting time ends, mix the above transfection reagent and the mRNA stock solution and let stand for 5 min. Slowly drop 50 μL of the mRNA-transfection reagent mixture into the culture plate, mix well, and culture at 37 °C for 24 h.

[0242] (2) Western blot assay

[0243] Discard the cell supernatant in the culture well plate of step (1), add 100 μl of 5× loading buffer to each well, add universal nuclease, lyse at room temperature for 5 min, and transfer to a 0.5 ml EP tube. Separate by 8-20% SDS-PAGE gel electrophoresis; after separation, perform membrane transfer to transfer the protein to a PVDF membrane; then, block the PVDF membrane in 3% bovine serum albumin, use the mouse serum immunized with the A35R, M1R, A29L, and B6R antigen proteins as the primary antibody, and use goat anti-mouse IgG-HRP as the secondary antibody, and incubate the PVDF membrane for 1 hour each in sequence; finally, develop the color with the developing solution.

[0244] The results are as Figure 2 shown, Figure 2 showing the expression of each antigen mRNA in the cells; as Figure 2 can be seen, A35R, M1R, A29L, and B6R in MPXV1 and MPXV2 are expressed in the cells, and the target bands can be detected. Among them, A35R and M1R in MPXV1 are fused into 1 molecule with a theoretical molecular weight of 39.9 kDa, and A29L and B6R are fused into 1 molecule with a theoretical molecular weight of 38.1 kDa; the 4 proteins of MPXV1 are expressed separately, the theoretical molecular weight of A35R is 20.1 kDa, the theoretical molecular weight of M1R is 27.3 kDa, the theoretical molecular weight of A29L is 12.6 kDa, and the theoretical molecular weight of B6R is 33.1 kDa. The apparent molecular weight shown in the Western blot assay is slightly higher than the theoretical molecular weight, which is caused by post-translational modification.

[0245] Example 3 Encapsulation of mRNA in Lipid Nanoparticles (LNP)

[0246] The cationic lipid (SM102, catalog number 2089251-47-6, Sinobioway Biotechnology Co., Ltd.), cholesterol (catalog number C3045-5G, Sigma-Aldrich Trading Co., Ltd.), phosphatidylcholine (DSPC, catalog number 5653PNA178, Avanti Polar Lipids, Inc.), and PEG lipid (DMG-PEG2000, catalog number 002005, Avanti Polar Lipids, Inc.) were mixed in a ratio of 50:38.5:10:1.5. Then, using a rapid nano-drug preparation instrument (Myanar (Shanghai) Instrument Technology Co., Ltd., INano L / 202101L24), the above-mentioned mRNA was encapsulated in liposomes, where A35R, M1R, A29L, and B6R of MPXV2 were encapsulated in a mass ratio of 1:1:1:1. After encapsulation, the buffer solution was changed to PBS by dialysis, and then concentrated using a 50 KD ultrafiltration concentrator tube. The concentrated mRNA-LNP had a concentration of about 0.2 mg / ml. The final product with qualified quality inspection was stored at 4°C until use. The quality inspection results are shown in Table 4:

[0247] Table 4

[0248]

[0249] Example 4 Experiment on Immunizing Mice with mRNA Vaccine

[0250] In this example, female mice of the BALB / c strain at 6-8 weeks of age were used for animal experiments; the experimental group was divided into an mRNA vaccine immunization group and a negative control group, and the negative control group was a PBS immunization group.

[0251] All mice in the mRNA vaccine immunization group of MPXV1 and MPXV2 were immunized with one dose of the mRNA vaccine prepared in Example 3 on day 0 and day 14 respectively, and the injection method was intramuscular injection on both sides of the thigh. The mice in the negative control group were injected with the same volume of PBS at the same time. The inoculation method was intramuscular injection, and the inoculation dose was 10 μg of mRNA vaccine or PBS per mouse each time. Mouse serum samples were collected on day 14 and day 28 respectively for testing the binding antibody titer and neutralizing antibody titer of the immune serum. Blood collection was carried out 2 days before immunization. The number of immunizations was two, and the immunization time and detection program time were as shown in the flowchart of Figure A in Figure 3 as shown in Figure A below.

[0252] (1) Detection of specific IgG antibody response

[0253] The antigens A35R, M1R, A29, and B6R were diluted to 10 μg / ml with ELISA coating buffer and added to a 96-well plate, followed by overnight coating at 4°C. After incubation with blocking buffer at room temperature for 1 hour, any uncoated protein was washed off using washing buffer. Serum was serially diluted to four concentrations and added to the wells, along with the corresponding serially diluted antibodies. After incubation, the serum was washed out and HRP-goat anti-mouse IgG was added for final optical density analysis in an ELISA reader.

[0254] The results are as Figure 3 shown in Figure B of 450 where the sera of mice in the MPXV1 group collected on day 28 were diluted at 1:2.57, 1:10,000, 1:50,000, and 1:200,000. When the dilution factor was 1:50,000, the OD 450 value of anti-A35R was still greater than 0.5; when the dilution factor was 1:10,000, the OD 450 value of anti-M1R was approximately 1.5; when the dilution factor was 1:10,000, the OD

[0255] value of anti-B6R was still greater than 1.5. This indicates 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 the A35R, M1R, A29, and B6R antigens. 3 ng / ml, 10 4 ng / ml, 10 5 -10 7 ng / ml, 10 4 ng / ml after the first immunization, and can reach 10 4 ng / ml, 10 5 ng / ml, 10 7 -10 8 ng / ml, 10 5 ng / ml after the second immunization. Moreover, the differences within the experimental group were small.

[0256] The results are shown in 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 increased after the second immunization.

[0257] (2) Detection of neutralizing antibody titer

[0258] To detect the content of neutralizing antibodies in the serum antibodies induced by the vaccine, the sera of mice 14 days after the second immunization were tested, and the test was commissioned by 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. Dilute the serum in a 4-fold gradient starting from 1:20 with serum-free medium, for a total of 6 gradients. Then take 120 μl of serum and mix it with an equal volume of vaccinia virus and incubate at 37 °C for 1 h. Add the incubated mixture to a Vero 96-well cell plate seeded the previous day (2×10 4 cells / mL), and after culturing at 37 °C for 72 h, stain by immunofluorescent plaque assay, read the plate and record the number of fluorescent spots in each well. Calculate the antibody titer of the serum according to the ratio of the number of fluorescent spots in the wells containing the test serum at different concentrations to the number of fluorescent spots in the positive control well without serum. Among them, PRNT 50 refers to the antibody concentration that can cause half of the maximum effect (i.e., 50% of the maximum inhibitory effect).

[0259] The results are shown in Figure 5 , The results showed that the cells in the negative cell wells without virus infection were normal and there were no obvious lesions. Obvious cytopathic effects appeared in the cells in the PBS group, the PRNT 50 value was 73, and there was no obvious neutralizing activity. Both the monkeypox MPXV1 and MPXV2 vaccines induced obvious neutralizing antibodies after immunization, and the neutralizing antibody titers were greater than 2000 after the second immunization. The PRNT 50 values were 3136 and 2065 respectively, and the neutralizing antibody titer after immunization with monkeypox MPXV1 was greater than that of MPXV2.

[0260] All the documents mentioned in the present invention are cited in this application as references, just as if each document was cited separately as a reference. 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 appended claims of this application.

Claims

1. A monkeypox virus multi-antigen fusion protein, characterized in that: The fusion protein has a structure as shown in Formula Ia from N-terminus to C-terminus: R1-L1-R2 Formula Ia 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; 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 a monkeypox virus A35R protein, the linker1 is a flexible peptide linker, and the M1R is a monkeypox virus M1R protein; 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, the linker2 is a flexible peptide linker, and the B6R is the monkeypox virus B6R protein; The amino acid sequence of the A35R is the amino acid sequence at positions 98 to 181 as shown in SEQ ID NO:16, the amino acid sequence of the M1R is the amino acid sequence at positions 1 to 250 as shown in SEQ ID NO:17, the amino acid sequence of the A29L is the amino acid sequence at positions 1 to 40 as shown in SEQ ID NO:18, and the amino acid sequence of the B6R is the amino acid sequence at positions 467 to 762 as shown in SEQ ID NO:

5.

2. The fusion protein according to claim 1, characterized in that The amino acid sequence of P1 is shown in SEQ ID NO:

1.

3. The fusion protein according to claim 1, characterized in that The amino acid sequence of P2 is shown in SEQ ID NO:

2.

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

5. The fusion protein according to claim 1, characterized in that The flexible peptide linker comprises 1-10 amino acids.

6. The fusion protein according to claim 1, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO:

5.

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 467 to position 762 as shown in SEQ ID NO: 5, 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 Type 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 a cell under appropriate conditions, wherein the nucleotide according to claim 8 is integrated into the genome of the cell, 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.

11. A use of the fusion protein according to claim 1, or the nucleotide according to claim 8, or the pharmaceutical composition according to claim 9, characterized in that: For the preparation of medicines, The medicament is used for treating and / or preventing monkeypox virus infection.

Images