A poxvirus single-stranded fusion immunogen, an immunogenic composition comprising the same, and their applications.

Abstract

This invention relates to a single-chain fusion immunogen against poxviruses (particularly monkeypoxvirus), an immunogenic composition comprising the same, and their applications. The single-chain fusion immunogen of this invention comprises four immunogens fused in tandem in a specific sequence: monkeypoxvirus A35, M1, B6, and A29 proteins or antigenic fragments thereof (or their derived peptides). It can elicit an immune response against both intracellular mature viral particles (IMV) and extracellular enveloped viral particles (EEV), thereby efficiently eliciting a specific immune protective effect against monkeypoxvirus. Furthermore, the poxvirus vaccine of this invention exhibits good safety, rapid response, and production capacity, demonstrating excellent prospects for clinical application.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a poxvirus single-chain fusion immunogen, an immunogenic composition containing the same, and their applications. Background Technology

[0002] Monkeypox is a viral zoonotic disease caused by the monkeypox virus, clinically manifested as fever, rash, and swollen lymph nodes. Since smallpox was eradicated in 1980 and vaccination against smallpox was subsequently discontinued, monkeypox has become the most important orpox virus in public health, making vaccine development an urgent priority.

[0003] The poxvirus genome consists of a large linear double-stranded DNA, nearly 200 kb in length, similar to vaccinia virus, and encodes up to 200 viral proteins. Monkeypox virus has the same morphology as other orthopoxviruses, being rounded brick-shaped or oval, measuring 200 nm × 250 nm, with a 30 nm outer membrane surrounding a homogeneous core. Monkeypox virus, along with smallpox virus, vaccinia virus, and vaccinia virus, are the four orthopoxviruses pathogenic to humans. They all contain soluble antigens, nucleoprotein antigens, and hemagglutinins, exhibiting essentially the same antigenic properties and cross-immunity. Monkeypox virus has two branches: one in West Africa and the other in the Congo Basin, with clear differences in epidemiology and clinical outcomes. The case fatality rate for monkeypox patients in the West African branch is approximately 3.6%, while the case fatality rate for those in the Congo Basin branch can reach 10.6%. Sequencing analysis of the monkeypox outbreak since May 2022 indicates that the virus belongs to the West African branch.

[0004] Poxviruses can produce two forms of infectious virions, called intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs), both of which contain a core and are surrounded by one (IMV) or two (EEV) lipid membranes. More than 20 viral proteins are distributed on the IMV membrane, most of which are involved in viral attachment and entry. However, EEVs possess a second outer membrane (enveloping membrane), which contains at least six unique envelope proteins. IMVs are the main progeny viral particles released after cell lysis, are structurally stable, and are responsible for mediating transmission between hosts, while EEVs are produced by exocytosis and are thought to transfer between cells or spread within the host.

[0005] Monkeypox virus (MPXV), along with smallpox virus (VARV), vaccinia virus (VACV), and cowpox virus (CPXV), belongs to the genus Orthopoxvirus in the family Poxviridae. Only these four viruses in the orthopoxvirus family can cause human infection. Early vaccines used to eradicate smallpox used live, unattenuated VAV strains produced from calf lymphocytes, such as the Dryvax vaccine; however, these vaccines are no longer produced due to efficacy and safety concerns. Smallpox vaccines offer cross-protection against monkeypox. Currently, the FDA has approved two vaccines for pre-exposure prophylaxis against orthopox, including monkeypox: JYNNEOS manufactured by Bavarian Nordic. TM The vaccine (also known as Imvamune or Imvanex) and Sanofi Pasteur are produced by Sanofi. Both vaccines are live attenuated vaccines originally used to prevent smallpox; among them, This is a second-generation vaccine, capable of replicating in the human body. Vaccination carries risks of encephalitis, myocarditis, and progressive smallpox infection. It is also unsuitable for infants, pregnant women, and individuals with weakened or compromised immune systems. (JYNNEOS) TM These are third-generation vaccines. Because they cannot replicate in the human body, their safety is improved compared to first- and second-generation vaccines, but their immunization efficacy is significantly reduced. It is worth noting that all of these vaccine products were approved after the eradication of smallpox, and therefore were not administered on a large scale in the general population. Their ability to control the spread of smallpox and monkeypox viruses and to eradicate these viruses remains to be determined.

[0006] Besides the limited target population and known side effects, attenuated live vaccines also present uncertainties and safety risks. First, there is the potential safety risk of vaccine strain mutations leading to a resurgence of virulence. Second, poxviruses, as large nucleoplasmic DNA viruses, have genomes ranging from 130-375 kbp and encoding up to 200 viral proteins, resulting in extremely complex antigenic components. The effective immunogen and mechanism of action of attenuated live vaccines are unclear, including several viral proteins with immunosuppressive functions, which could negatively impact the vaccine's immunogenicity and protective efficacy. These safety risks and uncertainties are particularly pronounced in immunocompromised populations such as the elderly and HIV carriers, limiting vaccination for these vulnerable groups. Furthermore, attenuated live vaccines also face challenges such as low production capacity and high costs. Therefore, given the severity of the pandemic and the various shortcomings of existing vaccines, there is an urgent need to leverage new technologies to develop a new generation of vaccines with clearly defined immunogenic components, well-defined mechanisms of action, and safe, effective, and accessible vaccines to aid in disease control.

[0007] Therefore, given the severity of the pandemic and the various shortcomings of existing vaccines, there is an urgent need to leverage new technologies to develop a new generation of vaccines with clear immunogenic components, well-defined mechanisms of action, safety, efficacy, and rapid availability, in order to aid in disease prevention and control.

[0008] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0009] Purpose of the invention

[0010] In view of the various drawbacks of existing vaccines, the purpose of this invention is to provide a monkeypox virus single-chain fusion immunogen that can efficiently stimulate a specific immune response against monkeypox virus (e.g., generate protective antibodies), related vaccine products, their preparation methods, and applications. In addition, vaccine products based on this single-chain fusion immunogen also have advantages such as safety, efficacy, clear immunogenic components and protective mechanisms, high production capacity, and low cost, thereby meeting the safety and production capacity requirements for large-scale vaccination of emergency populations.

[0011] Solution

[0012] To achieve the objectives of this invention, the following technical solutions are provided:

[0013] In a first aspect, the present invention provides a vaccinia virus single-chain fusion immunogen, the single-chain fusion immunogen comprising an amino acid sequence arranged in the pattern A35-C1-M1-C2-A35'-C3-A29-C4-B6, wherein:

[0014] A35 represents the monkeypox virus A35 protein or its antigenic fragment I, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with it and has the same or substantially the same immunogenicity.

[0015] A35' represents the monkeypox virus A35 protein or its antigenic fragment II, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with it and has the same or substantially the same immunogenicity.

[0016] M1 represents the monkeypox virus M1 protein or its antigenic fragment, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with it and has the same or substantially the same immunogenicity.

[0017] B6 represents the monkeypox virus B6 protein or an antigenic fragment thereof, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with it and has the same or substantially the same immunogenicity.

[0018] A29 represents the monkeypox virus A29 protein or an antigenic fragment thereof, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with it and has the same or substantially the same immunogenicity.

[0019] C1, C2, C3, and C4 are each independently either none or a concatenated sequence (GGGGS)n, where n is any integer between 1 and 10; and,

[0020] in,

[0021] A35 may be the same as or different from A35'.

[0022] C1, C2, C3, and C4 may be the same or different.

[0023] In some preferred embodiments, A35 represents an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence as shown in SEQ ID NO:1 obtained by substituting, deleting or adding one or more amino acids, which has the same or substantially the same immunogenicity as the amino acid sequence shown in SEQ ID NO:1.

[0024] And / or, A35' represents the amino acid sequence shown in SEQ ID NO:1, or the amino acid sequence shown in SEQ ID NO:1 plus a fragment extending 1-30 amino acids from it to the N-terminus of the A35 protein, or an amino acid sequence with the same or substantially the same immunogenicity obtained by substituting, deleting or adding one or more amino acids to the above amino acid sequence; preferably, A35' represents the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence with the same or substantially the same immunogenicity obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:2;

[0025] And / or, M1 represents the amino acid sequence shown in SEQ ID NO:3, or the amino acid sequence shown in SEQ ID NO:3 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity as it.

[0026] And / or, B6 represents the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence with the same or substantially the same immunogenicity as the amino acid sequence shown in SEQ ID NO:4 obtained by substituting, deleting or adding one or more amino acids.

[0027] And / or, A29 represents an amino acid sequence as shown in SEQ ID NO:5, or an amino acid sequence as shown in SEQ ID NO:5 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity as it.

[0028] More preferably, A35 represents the amino acid sequence shown in SEQ ID NO:1, A35' represents the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, M1 represents the amino acid sequence shown in SEQ ID NO:3, B6 represents the amino acid sequence shown in SEQ ID NO:4, and A29 represents the amino acid sequence shown in SEQ ID NO:5.

[0029] Preferably, C1, C2, C3, and C4 are all absent;

[0030] More preferably, the single-chain fusion immunogen comprises an amino acid sequence as shown in SEQ ID NO:6.

[0031] Preferably, the N-terminus of the single-chain fusion immunogen further includes a signal peptide sequence; optionally, the signal peptide sequence is shown in SEQ ID NO:9.

[0032] Preferably, the C-terminus of the single-chain fusion immunogen further includes a tag sequence; optionally, the tag is selected from at least one of the Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and SUMO tag, preferably the His tag.

[0033] In this invention, in order to take into account the immune protection effect against both EEV and IMV viral particles, a single-chain fusion immunogen containing neutralizing antigens A35 and B6 of EEV and neutralizing antigens M1 and A29 of IMV was designed. The four neutralizing antigens are encoded by the A35R, B6R, M1R and A29L genes of monkeypox virus, respectively, and they are homologous genes to vaccinia virus A33R, B5R, L1R and A27R, respectively.

[0034] In a second aspect, the present invention provides a method for preparing a single-chain fusion immunogen as described in the first aspect above, comprising the following steps:

[0035] The nucleotide sequence encoding the single-chain fusion immunogen described in the first aspect above is coupled with a Kozak sequence and a signal peptide coding sequence at the 5' end, and a histidine tag coding sequence and a stop codon at the 3' end. The nucleotide sequence is then cloned and expressed, and the correct recombinants are screened. The recombinants are then transfected into expression system cells for expression. The cell culture supernatant is collected, and the single-chain fusion immunogen is isolated from it.

[0036] In one feasible implementation of the above preparation method, the cells of the expression system are mammalian cells, insect cells, yeast cells, or bacterial cells;

[0037] Optionally, the mammalian cells are HEK293T cells, 293F series cells, or CHO cells; more preferably, the 293F series cells are HEK293F cells, Freestyle293F cells, or Expi293F cells.

[0038] Optionally, the insect cells are sf9 cells, Hi5 cells, sf21 cells, or S2 cells;

[0039] Optionally, the yeast cells are Pichia pastoris cells or yeast cells modified therefrom;

[0040] Optionally, the bacterial cells are Escherichia coli cells.

[0041] Thirdly, the present invention provides a polynucleotide that encodes the single-stranded fusion immunogen as described in the first aspect above.

[0042] In a specific implementation, the polynucleotide is a nucleotide sequence optimized with human codons, and can be DNA or mRNA;

[0043] Preferably, the polynucleotide is a DNA sequence as shown in SEQ ID NO:7;

[0044] Preferably, the polynucleotide is an mRNA sequence as shown in SEQ ID NO:8.

[0045] Fourthly, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the third aspect above, and optionally, at least one expression regulatory element operatively linked to the polynucleotide.

[0046] Fifthly, the present invention provides an expression vector comprising the nucleic acid construct as described in the fourth aspect above.

[0047] In a sixth aspect, the present invention provides a host cell wherein it is transformed or transfected with the polynucleotides as described in the third aspect above, the nucleic acid constructs as described in the fourth aspect above, or the expression vectors as described in the fifth aspect above.

[0048] In a seventh aspect, the present invention provides the use of the single-chain fusion immunogen as described in the first aspect above, the polynucleotide as described in the third aspect above, the nucleic acid construct as described in the fourth aspect above, the expression vector as described in the fifth aspect above, or the host cell as described in the sixth aspect above in the preparation of a medicament for the prevention and / or treatment of poxvirus infection.

[0049] Preferably, the poxvirus is selected from: monkeypox virus, smallpox virus, cowpox virus and / or vaccinia virus;

[0050] Optionally, the drug is a vaccine, preferably a recombinant protein vaccine; more preferably, the recombinant protein vaccine uses an adjuvant selected from the following: aluminum adjuvant, MF59 adjuvant, and MF59-like adjuvant;

[0051] Optionally, the vaccine may be in the form of a nasal spray, oral preparation, suppository, or parenteral preparation;

[0052] Preferably, the nasal spray is selected from aerosols, sprays, and powders;

[0053] Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated preparations, and ointments; more preferably, the tablets are sublingual tablets; more preferably, the granules are fine granules; more preferably, the powder is a powder; more preferably, the pills are small pills;

[0054] Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation; more preferably, the injectable preparation is a push-in preparation.

[0055] Eighthly, the present invention provides a vaccine or immunogenic composition comprising a single-stranded fusion immunogen as described in the first aspect above, a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above, an expression vector as described in the fifth aspect above, or a host cell as described in the sixth aspect above, and physiologically acceptable mediators, adjuvants, excipients, carriers, and / or diluents.

[0056] In some preferred embodiments, the vaccine or immunogenic composition is a monkeypox virus recombinant protein vaccine, which includes a single-chain fusion immunogen and an adjuvant as described in the first aspect above.

[0057] Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, and MF59-like adjuvant.

[0058] In some other preferred embodiments, the vaccine or immunogenic composition is a monkeypox virus DNA vaccine, comprising:

[0059] (1) Eukaryotic expression vectors; and

[0060] (2) Constructing a DNA sequence encoding the single-stranded fusion immunogen as described in the first aspect above into the eukaryotic expression vector, preferably the DNA sequence shown in SEQ ID NO:7;

[0061] Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

[0062] In some other preferred embodiments, the vaccine or immunogenic composition is a monkeypox virus mRNA vaccine, the mRNA vaccine comprising:

[0063] (I) an mRNA sequence encoding the single-stranded fusion immunogen as described in the first aspect above, preferably an mRNA sequence as shown in SEQ ID NO: 8; and

[0064] (II) Lipid nanoparticles.

[0065] In some other preferred embodiments, the vaccine or immunogenic composition is a monkeypox virus-virus vector vaccine, comprising:

[0066] (1) Viral backbone vector; and

[0067] (2) Constructing a DNA sequence encoding the single-stranded fusion immunogen as described in the first aspect above into the viral backbone vector, preferably a DNA sequence as shown in SEQ ID NO:7;

[0068] Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.

[0069] In a feasible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation;

[0070] Preferably, the nasal spray is selected from aerosols, sprays, and powders;

[0071] Preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments;

[0072] More preferably, the tablet is a sublingual tablet;

[0073] More preferably, the granules are fine granules;

[0074] More preferably, the powder is a granule;

[0075] More preferably, the pills are small pills;

[0076] Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation; more preferably, the injectable preparation is a push-in preparation.

[0077] In a ninth aspect, the present invention provides a method for preventing and / or treating poxvirus infection, the method comprising: administering to a subject in need a preventive and / or therapeutically effective amount of the following substances: a single-stranded fusion immunogen as described in the first aspect above, a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above, an expression vector as described in the fifth aspect above, a host cell as described in the sixth aspect above, and / or a vaccine or immunogenic composition as described in the eighth aspect above.

[0078] The "effective dose for prevention and / or treatment" may vary depending on the recipient, the organ involved, the symptoms, the method of administration, etc. It may be determined based on the doctor's judgment, taking into account factors such as the type of dosage form, the method of administration, the patient's age and weight, and the patient's symptoms.

[0079] Beneficial effects

[0080] The inventors of this application have designed a single-chain fusion immunogen against poxviruses (particularly monkeypoxviruses), comprising four antigenic epitopes fused in tandem in a specific order: (1) monkeypoxvirus A35 protein or an antigenic fragment thereof (or their derived peptides), (2) monkeypoxvirus M1 protein or an antigenic fragment thereof (or their derived peptides), (3) monkeypoxvirus B6 protein or an antigenic fragment thereof, and (4) monkeypoxvirus A29 protein or an antigenic fragment thereof; wherein M1 and A29 are neutralizing antigens specific to intracellular mature viral particles (IMV), and A35 and B6 are neutralizing antigens specific to extracellular enveloped viral particles (EEV); a vaccine containing both can elicit an immune response against the two infectious viral particles.

[0081] The single-chain fusion immunogen of the present invention contains two A35 proteins or their antigenic fragments that can form a stable intramolecular dimer, which is necessary for stimulating a class of neutralizing antibodies against the A35 antigen and also reverses the problem of immunogenicity loss when immunizing the A35 protein antigen alone.

[0082] Furthermore, the fusion immunogen of the present invention can induce balanced and high levels of specific antibodies against the four antigenic epitopes mentioned above; and, compared with immunization with the four antigen proteins alone, it can stimulate more efficient immune protection. Specifically, at the same immunization dose (e.g., 10 μg / dose in the examples), due to molecular weight differences, the effective number of moles of antigen protein when immunized with the antigen protein alone is several times that when immunized with the fusion immunogen (e.g., at an immunization dose of 10 μg / dose, it is approximately 3 times that of the fusion immunogen for the M1 antigen protein, approximately 2.4 times for the A35 antigen protein, approximately 2 times for the B6 antigen protein, and approximately 3.75 times for the A29 antigen protein). However, the fusion immunogen can still stimulate similar or better levels of specific and protective antibodies as when the antigen proteins are immunized alone, thus achieving unexpectedly more efficient immune protection.

[0083] Compared with existing poxvirus vaccines, vaccine products based on the single-chain fusion immunogen of this invention have the following advantages:

[0084] 1) Subunit vaccines have better safety, thus overcoming the safety issues of existing attenuated live virus vaccines; at the same time, compared with attenuated live virus vaccines, subunit vaccines have advantages such as lower production costs, faster response, and greater production capacity support; experimental verification shows that the poxvirus vaccine of this invention has good efficacy.

[0085] 2) The vaccine utilizes the antigenic sequence of monkeypox virus itself, which has high specificity against monkeypox virus. Existing live virus vaccines are basically developed based on vaccinia virus. Although vaccinia virus and monkeypox virus belong to the same family of poxviridae, there are still some differences in their neutralizing antigen sequences and antigenic epitopes. Therefore, their protective effect against monkeypox virus is still unclear. The vaccinia virus vaccine of this invention is developed based on the antigenic epitopes of monkeypox virus. Therefore, it has high specificity for the prevention and treatment of monkeypox virus.

[0086] 3) Monkeypox virus contains a wide variety of proteins, most of which cannot elicit an effective antiviral immune response and are therefore ineffective. In addition, some viral proteins have immunosuppressive effects. Existing live virus vaccines cannot remove the above-mentioned ineffective and harmful components, thus posing risks and uncertainties in vaccination. However, the poxvirus vaccine of this invention retains only four viral neutralizing antigens. Experimental data show that it can efficiently, stably and evenly elicit specific antibodies against these four antigens and can demonstrate a complete protective effect in mouse disease models.

[0087] Furthermore, compared with mixed immunogens containing multiple antigen proteins, the single-chain fusion immunogen of the present invention also has the following significant advantages:

[0088] 1) When using mixed immunogens for immunization, the differences in expression efficiency of different antigens on the mRNA technology platform, as well as the differences in molecular weight and stability of each antigen on the protein and mRNA technology platforms, will lead to differences in the degree of stimulation of the immune response by different antigens. Therefore, the bioavailability of each antigen will vary greatly, making it impossible to achieve a balance of immunity among different antigens. However, the single-chain fusion immunization principle of the present invention does not have the above problems.

[0089] 2) The single-chain fusion immunogen of the present invention is more conducive to productization. The construction of production cell lines, preclinical evaluation of safety, and product quality control can all be carried out for a single product, while the mixed immunization principle requires many times more work and cost.

[0090] Meanwhile, the B6 and A29 antigen fragments contained in the single-chain fusion immunogen of the present invention have been specifically modified. After this modification, the resulting antigen retains its antigenicity and neutralizing epitopes while avoiding unstable factors such as oligomerization caused by its own structure or sequence. This makes the constructed single-chain fusion immunogen express as a uniform monomer molecule as expected, meeting the requirements for protein uniformity in subsequent subunit vaccine development. Attached Figure Description

[0091] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0092] Figure 1 The gel filtration chromatography elution curve and SDS-PAGE identification results of the immunogenic protein AMAb2a expressed in Example 2 of the present invention are shown.

[0093] Figure 2 The gel filtration chromatography elution curves and SDS-PAGE identification results of the single immunogenic proteins A35(A), B6(B), A29(C) and M1(D) expressed in Example 2 of the present invention are shown.

[0094] Figure 3 The diagram shows the binding ability of the immunogenic protein AMAb2a, as described in Example 3 of this invention, to the neutralizing antibodies A27D7(A), 7D11(B), Anti-B6(C), and Anti-A29(D) for four antigenic epitopes (A35, M1, B6, and A29) as detected by ELISA; E represents the EC50 of AMAb2a binding to each antibody. 50 A list of values.

[0095] Figure 4This is a schematic diagram of the mouse immunization and challenge strategies used in Examples 4 and 7 of the present invention.

[0096] Figure 5 The table shows the specific binding antibody titers against the antigens A35(A), M1(B), B6(C), and A29(D) in the serum of each immunized mouse as detected in Example 5 of the present invention; wherein the vertical axis shows the endpoint antibody titer (Log10), and the horizontal axis shows each immunization group.

[0097] Figure 6 The table shows the neutralizing antibody titers against live VCV virus in the serum of each immunized mouse as detected in Example 6 of this invention; wherein, the vertical axis shows PRNT. 50 The (log10) value, with the horizontal axis representing each immune group.

[0098] Figure 7 This demonstrates the effects of each immunogen against high lethal doses (30LD5, 8 × 10⁸) as described in Example 7 of this invention. 5 The protective effect of PFU)VACV-WR virus intranasal inoculation on BALB / c mice is shown in the figure. The horizontal axis represents the number of days after the virus challenge, and the vertical axis represents the percentage change in body weight (A) and the survival rate (B) of the mice. The captions for each curve are shown on the right side of graph B. Detailed Implementation

[0099] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0100] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0101] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0102] Example 1: Immunogen Design

[0103] In this embodiment, as a representative example of the present invention, two A35 peptides, one M1 peptide, one B6 peptide, and one A29 peptide are tandemly arranged in a manner from the N-terminus to the C-terminus as A35-M1-A35-B6-A29. The resulting single-chain fusion peptide can form intramolecular dimerization of A35-A35, hereinafter referred to as AMAb2a, which represents the single-chain fusion immunogen of the present invention.

[0104] The following sequences are all from the monkeypox virus isolate MPXV_USA_2022_MA001, whose complete genome is available in GenBank at ON563414.3.

[0105] In AMAb2a, the two A35 peptides use different A35 protein regions: one is the S90-T181 region of the A35 protein (its amino acid sequence is shown in SEQ ID NO:1), and the other is the S64-T181 region of the A35 protein (its amino acid sequence is shown in SEQ ID NO:2). The amino acid sequence of the M1 peptide is shown in SEQ ID NO:3, the amino acid sequence of the B6 peptide is shown in SEQ ID NO:4, and the amino acid sequence of the A29 peptide is shown in SEQ ID NO:5. Therefore, the amino acid sequence of AMAb2a is shown in SEQ ID NO:6.

[0106] In constructing AMAb2a, for the needs of protein expression and purification, the inventors added a signal peptide sequence (as shown in SEQ ID NO:9) to the N-terminus of the AMAb2a amino acid sequence as shown in SEQ ID NO:6, and added 8 histidine tags to its C-terminus, thereby forming the complete construct shown in SEQ ID NO:10:

[0107] AMAb2a complete construct (SEQ ID NO:10)

[0108] The underlined portion at the beginning is the signal peptide sequence. The first bold part is the first A35 peptide sequence (S89-T180), the second underlined part is the M1 peptide sequence (A2-G181), the second bold part is the second A35 peptide sequence (S63-T180), the italic part is the B6 peptide sequence (T20-A128), the third bold part is the A29 peptide sequence (M1-N43), and the last underlined portion is the histidine tag sequence.

[0109] In addition, for comparison, this embodiment also designed expression constructs for single antigens A35, M1, A29, and B6 peptides, whose amino acid sequences are as follows:

[0110] The amino acid sequence of the A35 peptide construct (SEQ ID NO:11):

[0111] The italicized part at the beginning is the signal peptide sequence, the bold part in the middle is the A35 peptide sequence, and the underlined part at the end is the histidine tag sequence for purification.

[0112] The amino acid sequence of the M1 peptide construct (SEQ ID NO:12):

[0113] The italicized portion at the beginning is the tag sequence for purification, and the bold portion at the end is the M1 peptide sequence.

[0114] The amino acid sequence of the A29 peptide construct (SEQ ID NO:13):

[0115] The bold part is the A29 peptide sequence, and the italic part is the purification tag sequence.

[0116] The amino acid sequence of the B6 peptide construct (SEQ ID NO:14):

[0117] The italicized portion at the beginning is the signal peptide sequence, the bold portion in the middle is the B6 peptide sequence, and the underlined portion at the end is the histidine tag for purification.

[0118] Example 2: Expression and purification of immunogenic proteins

[0119] Expression and purification of the construct AMAb2a

[0120] The amino acid sequence of the construct AMAb2a designed in Example 1 was optimized with human codons to obtain the nucleotide sequence encoding AMAab2, as shown in SEQ ID NO:15. A Kozak sequence (GCCACC) was added to the 5' end of the nucleotide sequence and a translation stop codon was added to the 3' end. Then, a DNA fragment was artificially synthesized and cloned into the EcoRI and XhoI restriction sites of the pCAGGS vector to obtain the expression plasmid of the construct AMAb2a.

[0121] The AMAb2a expression plasmid was transfected into 293F cells for in vitro recombinant expression. Five to seven days post-transfection, cell supernatants containing the expressed immunogenic protein were collected. Protein purification was then performed; specifically, the cell supernatant containing the immunogenic protein was subjected to HisTrap chromatography. TM The protein was crudely purified by HP (GE) chromatography, and then further purified by Superdex 200 10 / 300GL (GE) gel filtration chromatography. Finally, the purity and molecular weight of the protein were identified by SDS-PAGE.

[0122] Elution curves of gel filtration chromatography and identification results of SDS-PAGE are as follows: Figure 1 As shown, Figure 1 This indicates that after two-step purification, high-purity AMAb2a protein with a molecular weight of ~75kDa can be obtained, which is in line with expectations.

[0123] Expression and purification of single immunogens A35, M1, A29 and B6

[0124] The amino acid sequences of the single antigenic peptides A35, M1, A29, and B6 designed in Example 1 were optimized using codons in E. coli (M1) and human (A35, B6, A29) to obtain their encoding nucleotide sequences, as shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively. Translation stop codons were added to the 3' end of these nucleotide sequences. Then, these DNA fragments were artificially synthesized and cloned into pCAGGS (A35, B6, A29) or pET-28a expression vectors (M1) to obtain expression plasmids for A35, M1, A29, and B6.

[0125] The expression and purification process for A35, B6, and A29 was consistent with that for AMAb2a. The elution curves from gel filtration chromatography and the SDS-PAGE identification results are as follows: Figure 2 As shown in A, 2B, and 2C; Figure 2A, 2B, and 2C indicate that high-purity A35, B6, and A29 proteins can be obtained after two-step purification. SDS-PAGE analysis of A35 protein showed a monomeric molecular weight of ~15 kDa, and the elution peak from gel filtration chromatography corresponded to a molecular weight of ~30 kDa, indicating that it exists in a dimer form. The ability of A35 to form a dimer has been confirmed in the structure of its poxvirus homolog A33. Purification of B6 protein using a Hiload 16 / 60 Superdex 200PG gel electrophoresis showed an elution peak and SDS-PAGE analysis indicating a molecular weight of ~40 kDa, existing as a monomer in solution. Purification of A29 protein using a Hiload 16 / 60 Superdex 200PG gel electrophoresis showed an SDS-PAGE analysis indicating a molecular weight of ~20 kDa, with two bands, possibly due to different glycosylation forms; simultaneously, the elution peak corresponded to a molecular weight greater than 70 kDa, suggesting that A29 protein exists in a homogeneous multimer form in solution. The ability of A29 to form multimers within the structure of its vaccinia virus homolog A27 has been confirmed. The expression and purification results described above are in line with expectations.

[0126] The expression and purification process of M1 is as follows: The expression plasmid of M1 was expressed using an *E. coli* system. The resulting inclusion bodies were refolded in vitro using an arginine dilution method to obtain the active protein M1. Then, the refolded M1 protein was purified using a Superdex 200 100 / 300GL gel filtration chromatography column. Finally, the protein purity and molecular weight were identified by SDS-PAGE. The elution curve of gel filtration chromatography and the results of SDS-PAGE identification are shown below. Figure 2 As shown in D. Figure 2 As shown in D, after the above-mentioned gel filtration chromatography purification, high-purity M1 protein can be obtained; its peak position and SDS-PAGE both show ~25kDa, which is in line with expectations.

[0127] Example 3: Antigenicity detection of immunogens

[0128] To detect the exposure status and antigenicity of each antigenic epitope of the single-chain fusion immunogen of the present invention, in this embodiment, the binding ability of the immunogen proteins expressed and purified in Example 2 (including the single-chain fusion immunogen protein AMAb2a and the individual antigen proteins A35, M1, A29 and B6) to the neutralizing antibodies of each epitope was detected by ELISA. Among them, the 7D11 and A27D7 antibodies are neutralizing antibodies against vaccinia virus antigens L1 and A33, respectively (see Hua-Poo Su et al., Structural basis for the binding of the neutralizing antibody, 7D11, to the poxvirus L1 protein, Virology 368 (2007) 331–341; and Michael H. Matho et al., Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer, PLOS Pathogens | DOI: 10.1371 / journal.ppat.1005148 September). (1, 2015), data show that they can cross-recognize the M1 and A35 antigens of monkeypox virus, with antibody A27D7 able to recognize the dimeric epitope of A35; antibodies against A29 and B6 antigens were obtained in our laboratory through screening immunized mice.

[0129] The results are as follows Figure 3 As shown.

[0130] Depend on Figure 3 It can be seen that the fusion immunogen AMAb2a exhibits similar binding affinity to the A35 epitope antibody (A27D7), M1 epitope antibody (7D11), B6 ​​epitope antibody (Anti-B6), and A29 epitope antibody (Anti-A29) as to the individual A35, M1, B6, and A29 antigen proteins, as shown in the EC50 of the ELISA binding curves. 50 The values ​​are all at similar levels.

[0131] The above data show that all four antigenic epitopes on the single-chain fusion immunogen AMAb2a of the present invention can be well exposed, exhibiting high antigenicity.

[0132] Example 4: Mouse Immunization Experiment

[0133] To verify the immunoprotective efficacy of the single-chain fusion immunogen of the present invention, the inventors reacted each immunogen protein obtained in Example 2 with AddaVax. TM Adjuvant mixture emulsification, according to Figure 4The strategy was to immunize mice. The replicable vaccinia virus strain Tian Tan (VACV-VTT) was used as a control group for the attenuated live vaccine.

[0134] In the immunization experiment, all mice used were female BALB / c mice, aged 6-8 weeks, with an average weight of 15-20g. Each experimental group used 8 mice. Figure 5 As shown, mice were immunized on days 0, 21 and 42, for a total of 3 times, with a dose of 10 μg / mouse each time; the vaccination was administered by intramuscular injection, with 50 μL injected into each leg in the thigh.

[0135] VACV-VTT mice were immunized using a tail root scratch method, specifically, immunization was performed on day 0, with a total of one immunization, and the immunization dose was 10. 7 PFU / each.

[0136] Two days before the second and third immunizations and the challenge (i.e., days 19, 40, and 61), blood was collected from the orbital sinus of mice in all groups. After allowing the blood to clot, mouse serum was obtained by centrifugation at 1500 rpm for 10 minutes. The mouse serum was immediately aliquoted and stored at -80°C for subsequent ELISA detection of specific antibody titers and determination of live virus neutralizing capacity.

[0137] Example 5: ELISA assay for detecting vaccine-induced specific antibody titers

[0138] Each immunogenic protein used for mouse immunization (i.e., single antigen proteins A35, M1, A29, and B6) was diluted to 2 μg / mL with ELISA coating buffer (Solepro, C1050). 100 μL of the diluted immunogenic protein was added to each well of a 96-well ELISA plate (Corning, 3590), and the plate was incubated overnight at 4°C. The coating buffer was discarded, and PBS was added to wash away any residual coating buffer. 100 μL of ELISA blocking buffer (10% skim milk powder prepared with PBST) was added, and the plate was incubated at room temperature for 1 hour for blocking. During the blocking process, the immunized mouse sera obtained in Example 4 were diluted with ELISA blocking buffer, starting from 200-fold and serially diluted 3-fold, with 11 dilutions per sample. After blocking, the blocking buffer was removed, and 100 μL of immunized mouse sera diluted 10-fold with the blocking buffer was added to the ELISA plate. The plate was incubated at room temperature for 1 hour, followed by washing three times with PBST. Then, HRP-labeled goat anti-mouse secondary antibody (Abcam, ab6789) diluted 1:4000 with the blocking buffer was added, and the plate was incubated at room temperature for 1 hour. The plate was then washed 5-6 times with PBST, and TMB chromogenic buffer was added for color development. The reaction was terminated by adding 2M hydrochloric acid after an appropriate reaction time. OD450 readings were detected using a microplate reader. An OD450 value greater than 2.5 times that of the negative control was considered positive. The highest dilution of the serum that was considered positive was defined as the serum antibody titer. When the reaction value at the lowest dilution factor is still less than 2.5 times that of the negative control, the potency of the sample is defined as half of the lowest dilution factor, i.e., Log10 = 1.

[0139] The serum antibody titers against each antigenic epitope in mice immunized with each immunogenic protein are as follows: Figure 5 As shown; by Figure 5 It can be seen that the single-chain fusion antigen AMAb2a of the present invention can uniformly stimulate specific antibodies against four antigenic epitopes and has good immunogenicity; the ability of AMAb2a to stimulate specific antibodies against the four epitopes is higher than that of the live virus immunization group; at the same time, AMAb2a greatly reverses the problem of poor immunogenicity when A35 is immunized alone; both the single immunization and the fusion immunogen immunization are 10ug / dose, but due to the difference in molecular weight, the effective number of antigen protein moles in the single immunization is about 3 times (M1), 2.4 times (A35), 3.75 times (A29) or 2 times (B6) of the fusion immunogen immunization, but the fusion immunogen AMAab2 can still stimulate a similar level of specific antibody as the single immunization.

[0140] Example 6: Determination of the neutralizing capacity of live VCV virus in immune serum

[0141] Vacciniavirus (VACV) is the model virus of the genus Poxvirus. Due to the high homology among the major immunogens of the genus Poxvirus, VACV, with its lower biosafety level and ease of handling, is internationally used for evaluating the cellular-level neutralizing capacity of other poxvirus vaccines and their protective efficacy in mouse models. Therefore, in this embodiment, the internationally used internationally adopted mouse strain of VACV (Western Reserve, VACV-WR) was used to determine the cellular-level live virus neutralizing capacity of various immunogenic proteins.

[0142] The immunized mouse serum obtained in Example 4 was diluted with DMEM containing 2% inactivated serum, serially diluted 2-fold starting from 20-fold, with 10 dilutions per sample. The VACV-WR virus was also diluted to 500 PFU / mL using the same diluent. 200 μL of the diluted immunized mouse serum (the diluent without immunized mouse serum was used as a control well) was mixed with 200 μL of the diluted virus solution and incubated at 37°C for 1 hour. Vero cells were seeded in 12-well plates one day in advance, with the optimal density being approximately 95% on the second day. The culture medium in the 12-well plates was discarded, and residual culture medium was washed away with PBS. The incubated serum-virus mixture was then added to the 12-well plates, and the plates were incubated at 37°C for 2 hours. After infection, the virus-serum mixture was removed, residual virus was washed away with PBS, and the prepared carboxymethyl cellulose-DMEM mixture (2% carboxymethyl cellulose and 2×DMEM mixed in a 1:1 ratio) was added. The plates were incubated at 37°C for 48-60 hours. After observing obvious CPE under a microscope, 4% paraformaldehyde fixative was added, and the mixture was fixed at room temperature for 2 hours. Then, crystal violet staining was performed, and the CPE counts were performed.

[0143] PRNT 50 The calculation method is as follows: Wells containing only the virus are used as control wells. The number of empty plaques after counting all experimental wells is divided by the number of empty plaques in the control wells to obtain the virus inhibition rate of serum for each experimental well. Then, the PRNT is calculated using the log(inhibitor) vs. normalized response -- Variable slope formula in GraphPad software. 50 .

[0144] See results Figure 6 .

[0145] Because the live virus neutralization assay used the VCV-WR strain, which produces almost no EEV, our neutralization assays all used IMV virus particles. Since A35 and B6 are not present on IMV virus particles, we did not test the neutralizing capacity of the serum from the A35 and B6 immunized groups.

[0146] like Figure 6As shown, the A29, M1, AMAb2a and live vaccine immunization groups can all produce neutralizing antibodies against IMV virus particles. Among them, AMAb2a has a significantly higher neutralizing antibody elicitation capacity than the live vaccine VCV-VTT and A29 immunization groups.

[0147] Example 7: Virus Challenge Protection Experiment

[0148] like Figure 5 As shown, in Example 4, mice that underwent three immunizations were given a high lethal dose of 8 × 10⁻⁶ on day 63, three weeks after the third immunization. 5 The VAV-WR virus intranasal challenge experiment with PFU / mouse (30LD50) was used to evaluate the protective effect of the vaccine in animal models. The percentage change in body weight and survival rate of mice in each challenge group are shown in the figure. Figure 7 .

[0149] Figure 7 The results show that:

[0150] 1) The single antigen A35 immunization group and the single antigen A29 immunization group had no protective effect against VCV-WR virus infection in mice. All mice died 6 days after challenge. This indicates that although the two antigens can stimulate specific antibody levels after three immunizations (among which, the A29 single antigen stimulated A29 specific antibody levels were higher than those in the AMAb2a group), they still cannot provide comprehensive protection against lethal VCV-WR infection.

[0151] 2) In the single antigen M1 immunization group, the survival rate of mice was 87.5% (7 / 8), but the mice experienced significant weight changes; these results indicate that although M1 immunization alone can elicit high levels of specific antibodies and has superior cellular neutralization capacity in all immunization groups, it still cannot provide comprehensive protection against lethal VCV-WR infection.

[0152] 3) In the single antigen B6 immunization group and the attenuated live vaccine immunization group, although the survival rate of mice was 100%, the mice experienced significant weight changes on days 3-6 after challenge (VACV-VTT group) or days 3-8 (B6 immunization group).

[0153] 4) Compared with the above-mentioned immunization group, the single-chain fusion immunogen AMAb2a of this application has a 100% protective effect against VACV-WR-infected mice, and no significant weight change was observed in the mice.

[0154] The above results indicate that the single-chain fusion immunogen AMAb2a of the present invention exhibits excellent protective effects in mouse models, and therefore has excellent application prospects in the preparation of poxvirus vaccines to prevent poxvirus infection, including monkeypoxvirus.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

[0156] The sequences involved in this application are:

[0157] SEQ ID NO:1 (Amino acid sequence of the first A35 peptide in AMAb2a)

[0158] STTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT

[0159] SEQ ID NO:2 (Amino acid sequence of the second A35 peptide in AMAb2a)

[0160] SANKAAITDSAVAVAAASSTHRKVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT

[0161] SEQ ID NO:3 (Amino acid sequence of M1 peptide in AMAb2a)

[0162] AAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSKGNCAIKALMQLTTKATTQIAPRQVAG

[0163] SEQ ID NO:4 (Amino acid sequence of B6 peptide in AMAb2a)

[0164] TCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNA

[0165] SEQ ID NO:5 (Amino acid sequence of peptide A29 in AMAb2a)

[0166] MDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDN

[0167] SEQ ID NO:6 (Amino acid sequence of AMAb2a)

[0168] STTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCTAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGSANKAAITDSAVAVAAASSTHRKVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCTTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAMDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDN

[0169] SEQ ID NO:7 (DNA sequence encoding AMAb2a as shown in SEQ ID NO:6)

[0170]

[0171] SEQ ID NO:8 (encoding the mRNA sequence of AMAab2 as shown in SEQ ID NO:6)

[0172]

[0173] SEQ ID NO:9 (Amino acid sequence of the signal peptide)

[0174] METDTLLLWVLLLWVPGSTG

[0175] SEQ ID NO:10 (Amino acid sequence of the complete AMAb2a construct in Example 1)

[0176] METDTLLLWVLLLWVPGSTGSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCTAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGSANKAAITDSAVAVAAASSTHRKVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCTTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAMDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNHHHHHHHH

[0177] SEQ ID NO:11 (Amino acid sequence of the single antigen A35 peptide in Example 1)

[0178] METDTLLLWVLLLWVPGSTGSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCTHHHHHH

[0179] SEQ ID NO:12 (Amino acid sequence of the single antigen M1 peptide in Example 1)

[0180] MGSSHHHHHHSSGLVPRGSHMGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAG

[0181] SEQ ID NO:13 (Amino acid sequence of the single antigen A29 peptide in Example 1)

[0182] MDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKFEQIEKSSKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRHPYELVPRGSGWSHPQFEKGGGSGGGSGGSAWSHPQFEK

[0183] SEQ ID NO:14 (Amino acid sequence of the single antigen B6 peptide in Example 1)

[0184] MGAGATGRAMDGPRLLLLLLLGVSLGGAASTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHHHHHHHH

[0185] SEQ ID NO:15 (nucleotide sequence encoding the complete AMAb2a construct)

[0186]

[0187] SEQ ID NO:16 (nucleotide sequence encoding a single antigen A35 peptide (i.e., SEQ ID NO:11))

[0188] ATGGAAACAGACACCCTGCTGCTGTGGGTCCTGCTGCTCTGGGTGCCTGGCAGCACCGGCAGCACCACCCAGTACGACCACAAAGAGAGCTGCAACGGCCTGTACTACCAAGGCTCTTGTTACATCCTGCACAGCGACTATAAGAGCTTCGAGGATGCTAAGGCCAATTGCGCCGCCGAGAGCAGCACACTGCCTAACAAGTCCGACGTGCTGACCACCTGGCTGATCGACTACGTGGAAGATACATGGGGAAGCGACGGCAACCCCATCACAAAAACCACAAGCGATTATCAGGACAGCGACGTGTCTCAGGAGGTTAGAAAGTACTTTTGCACCCACCACCACCACCACCAT

[0189] SEQ ID NO:17 (nucleotide sequence encoding a single antigen M1 peptide (i.e., SEQ ID NO:12))

[0190] atgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggcagccatatgggagcagctgcgtcaatacaaacaactgtaaacaccctgagcgaacgtattagctccaaacttgagcaagaggcaaacgcgagcgcgcaaacgaaatgcgatattgagatcggcaacttctatatccgccaaaatcacggttgtaatattaccgtcaagaacatgtgcagcgcggacgcggacgcgcagctggacgccgttttgtctgcagcgaccgaaacctattccggtctgaccccggagcagaaagcgtacgttccggctatgttcaccgcagcactcaatatccaaaccagcgtcaataccgttgttcgtgattttgaaaattacgtgaagcagacgtgcaactcctcggcggtggtggataacaaactgaagatccaaaacgtgattatcgacgaatgttacggcgctccgggttctccgaccaacttggagtttatcaacactggcagcagcaaaggcaactgcgctattaaggcgctgatgcagctgactacaaaggcgaccacgcagatcgccccacgtcaggtggccggt

[0191] SEQ ID NO:18 (nucleotide sequence encoding a single antigen A29 peptide (i.e., SEQ ID NO:13))

[0192] atggatggtaccctgttccccggcgatgacgacttggccatccccgcgaccgagtttttctccactaaggcggcgaagaacccggagacaaagcgggaggccatcgtcaaggcgtacggggacgacaatgaggagacgctgaagcagcgtctcacgaacttggagaagaaaattacaaacattacgaccaagttcgagcagattgagaagAgcAgcaagagaaacgatgaagtcctcttccggctggagaatcacgcagagacactaagggctgcgatgatttctctagccaagaagatagacgtccagacggggcgccatccatacgagctggtgcctagaggctctggctggtcccacccccagttcgaaaagggcggcggcagcggcggaggaagcggcggcagcgcctggagccaccctcagttcgagaag

[0193] SEQ ID NO:19 (nucleotide sequence encoding a single antigen B6 peptide (i.e., SEQ ID NO:14))

[0194] ATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCCGCTAGCACCTGCACCGTGCCCACCATGAACAACGCCAAGCTGACCAGCACCGAGACAAGCTTCAACGACAAGCAGAAGGTGACCTTCACCTGCGACAGCGGCTACCACAGCCTGGACCCCAACGCCGTGTGCGAGACAGACAAGTGGAAGTACGAGAACCCCTGCAAGAAGATGTGCACCGTGAGCGACTACGTGAGCGAGCTGTACGACAAGCCCCTGTACGAGGTGAACAGCACCATGACCCTGAGCTGCAACGGCGAGACAAAGTACTTCCGGTGCGAGGAGAAGAACGGCAACACCAGCTGGAACGACACCGTGACCTGCCCCAACGCCGAGTGCCAGCCCCTGCAGCTGGAGCACGGCAGCTGCCAGCCCGTGAAGGAGAAGTACAGCTTCGGCGAGTACATGACCATCAACTGCGACGTGGGCTACGAGGTGATCGGCGTGAGCTACATCAGCTGCACCGCCAACAGCTGGAACGTGATCCCCAGCTGCCAGCAGAAGTGCGACATCCCCAGCCTGAGCAACGGCCTGATCAGCGGCAGCACCTTCAGCATCGGCGGCGTGATCCACCTGAGCTGCAAGAGCGGCTTCACCCTGACCGGCAGCCCCAGCAGCACCTGCATCGACGGCAAGTGGAACCCCATCCTGCCCACCTGCGTGCGGAGCAACGAGGAGTTCGACCCCGTGGACGACGGCCCCGACGACGAGACAGACCTGAGCAAGCTGAGCAAGGACGTGGTGCAGTACGAGCAGGAGATCGAGAGCCTGGAGGCCACCTACCACcatCACCACCACCATCATCAC

Claims

1. A poxvirus single-stranded fusion immunogen, characterized in that, The single-chain fusion immunogen has an amino acid sequence arranged in the pattern A35-C1-M1-C2-A35'-C3-B6-C4-A29, wherein: A35 represents the monkeypox virus A35 protein, wherein A35 represents the amino acid sequence shown in SEQ ID NO:

1. A35' represents the amino acid sequence shown in SEQ ID NO:

2. M1 represents the monkeypox virus M1 protein, wherein M1 represents the amino acid sequence shown in SEQ ID NO:

3. B6 represents the monkeypox virus B6 protein, wherein B6 represents the amino acid sequence shown in SEQ ID NO:

4. A29 represents the monkeypox virus A29 protein, wherein A29 represents the amino acid sequence shown in SEQ ID NO:

5. C1, C2, C3, and C4 are either none or a concatenated sequence (GGGGS)n, where n is any integer between 1 and 10.

2. The poxvirus single-chain fusion immunogen according to claim 1, characterized in that, C1, C2, C3, and C4 are all absent.

3. The poxvirus single-stranded fusion immunogen according to claim 1, characterized in that, The single-chain fusion immunogen has the amino acid sequence shown in SEQ ID NO:

6.

4. A poxvirus single-stranded fusion immunogen, characterized in that, From the N-terminus to the C-terminus are: a signal peptide sequence and the poxvirus single-chain fusion immunogen as described in any one of claims 1-3.

5. The poxvirus single-stranded fusion immunogen according to claim 4, characterized in that, The signal peptide sequence is shown in SEQ ID NO:

9.

6. A poxvirus single-stranded fusion immunogen, characterized in that, From the N-terminus to the C-terminus are: the poxvirus single-stranded fusion immunogen as described in any one of claims 1-3, and the tag sequence.

7. The poxvirus single-stranded fusion immunogen according to claim 6, characterized in that, The tag is selected from at least one of the following: Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and SUMO tag.

8. The poxvirus single-stranded fusion immunogen according to claim 6, characterized in that, The label is the His label.

9. A method for preparing a single-chain fusion immunogen, comprising the following steps: The nucleotide sequence encoding the single-chain fusion immunogen as described in any one of claims 1-3 is coupled with a Kozak sequence and a signal peptide coding sequence at the 5' end, and a histidine tag coding sequence and a stop codon at the 3' end. The cloning and expression are performed, the correct recombinants are screened, and then the recombinants are transfected into expression system cells for expression. The cell culture supernatant is collected, and the single-chain fusion immunogen is isolated from it.

10. The preparation method according to claim 9, characterized in that: The expression system cells are mammalian cells, insect cells, yeast cells, or bacterial cells.

11. The preparation method according to claim 10, characterized in that, The mammalian cells are HEK293T cells, 293F series cells, or CHO cells.

12. The preparation method according to claim 11, characterized in that, The 293F series cells are HEK293F cells, Freestyle293F cells, or Expi293F cells.

13. The preparation method according to claim 10, characterized in that, The insect cells are sf9 cells, Hi5 cells, sf21 cells, or S2 cells.

14. The preparation method according to claim 10, characterized in that, The yeast cells are Pichia pastoris cells or Pichia pastoris cells modified from them.

15. The preparation method according to claim 10, characterized in that, The bacterial cells are Escherichia coli cells.

16. A polynucleotide encoding a single-stranded fusion immunogen as described in any one of claims 1-8.

17. The polynucleotide according to claim 16, characterized in that: The polynucleotide is DNA or mRNA.

18. The polynucleotide according to claim 16, characterized in that: The polynucleotide is a DNA sequence as shown in SEQ ID NO:

7.

19. The polynucleotide according to claim 16, characterized in that: The polynucleotide is an mRNA sequence as shown in SEQ ID NO:

8.

20. A nucleic acid construct comprising the polynucleotide as described in any one of claims 16 to 19.

21. The nucleic acid construct according to claim 20, characterized in that: It also includes at least one expression regulatory element operatively linked to the polynucleotide.

22. An expression vector comprising the nucleic acid construct as described in claim 20 or 21.

23. A host cell wherein it is transformed or transfected with a polynucleotide as described in any one of claims 16 to 19, a nucleic acid construct as described in claim 20 or 21, or an expression vector as described in claim 22.

24. The use of the single-chain fusion immunogen as described in any one of claims 1-8, the polynucleotide as described in any one of claims 16-19, the nucleic acid construct as described in claim 20 or 21, the expression vector as described in claim 22, or the host cell as described in claim 23 in the preparation of a medicament for preventing poxvirus infection; The poxvirus is selected from: monkeypoxvirus and / or vaccinia virus; The drug in question is a vaccine.

25. The application according to claim 24, characterized in that, The vaccine is a recombinant protein vaccine.

26. The application according to claim 25, characterized in that, The recombinant protein vaccine uses an adjuvant selected from the following: aluminum adjuvant, MF59 adjuvant, and MF59-like adjuvant.

27. The application according to claim 24, characterized in that, The vaccine is in the form of a nasal spray, oral preparation, or suppository.

28. The application according to claim 27, characterized in that, The nasal spray is selected from aerosols, sprays, and powders.

29. The application according to claim 27, characterized in that, The oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated formulations, and ointments.

30. The application according to claim 29, characterized in that, The tablets mentioned are sublingual tablets.

31. The application according to claim 29, characterized in that, The granules are fine granules.

32. The application according to claim 29, characterized in that, The powder is a granule.

33. The application according to claim 29, characterized in that, The pills mentioned are small pills.

34. The application according to claim 24, characterized in that, The vaccine is in the form of a parenteral preparation.

35. The application according to claim 34, characterized in that, The parenteral preparation is a transdermal preparation.

36. The application according to claim 34, characterized in that, The parenteral preparation is an ointment or plaster.

37. The application according to claim 34, characterized in that, The parenteral preparation is a topical liquid.

38. The application according to claim 34, characterized in that, The parenteral preparation is an injectable preparation.

39. The application according to claim 38, characterized in that, The injectable formulation is a push-in formulation.

40. An immunogenic composition comprising a single-stranded fusion immunogen as claimed in any one of claims 1-8, a polynucleotide as claimed in any one of claims 16-19, a nucleic acid construct as claimed in claim 20 or 21, an expression vector as claimed in claim 22, or a host cell as claimed in claim 23, and a physiologically acceptable medium, adjuvant, excipient, carrier, and / or diluent.

41. A vaccine comprising a single-stranded fusion immunogen as claimed in any one of claims 1-8, a polynucleotide as claimed in any one of claims 16-19, a nucleic acid construct as claimed in claim 20 or 21, an expression vector as claimed in claim 22 or a host cell as claimed in claim 23, and a physiologically acceptable medium, adjuvant, excipient, carrier and / or diluent.

42. The vaccine according to claim 41, which is a monkeypox virus recombinant protein vaccine, comprising the single-chain fusion immunogen and adjuvant as described in any one of claims 1-8.

43. The vaccine according to claim 42, wherein the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, and MF59-like adjuvant.

44. The vaccine according to claim 41, wherein it is a monkeypox virus DNA vaccine, the DNA vaccine comprising: (1) Eukaryotic expression vector; and (2) Construct a DNA sequence encoding the single-stranded fusion immunogen as described in any one of claims 1-8 into the eukaryotic expression vector.

45. The vaccine according to claim 44, characterized in that, (2) The DNA sequence encoding the single-stranded fusion immunogen is the DNA sequence shown in SEQ ID NO:

7.

46. ​​The vaccine according to claim 44, characterized in that, (1) The eukaryotic expression vectors mentioned are selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

47. The vaccine according to claim 41, wherein it is a monkeypox virus mRNA vaccine, the mRNA vaccine comprising: (I) An mRNA sequence encoding the single-stranded fusion immunogen as described in any one of claims 1-8; and (II) Lipid nanoparticles.

48. The vaccine according to claim 47, characterized in that, In (I), the mRNA sequence encoding the single-stranded fusion immunogen is the mRNA sequence shown in SEQ ID NO:

8.

49. The vaccine according to claim 41, which is a monkeypox virus-virus vector vaccine, comprising: (1) Viral backbone vector; and (2) Constructing a DNA sequence encoding a single-stranded fusion immunogen as described in any one of claims 1-8 into the viral backbone vector.

50. The vaccine according to claim 49, characterized in that, In (2), the DNA sequence encoding the single-stranded fusion immunogen is the DNA sequence shown in SEQ ID NO:

7.

51. The vaccine according to claim 49, characterized in that, In (1), the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.

52. The vaccine according to any one of claims 44-51, characterized in that, The vaccine is in the form of a nasal spray, oral preparation, or suppository.

53. The vaccine according to claim 52, characterized in that, The nasal spray is selected from aerosols, sprays, and powders.

54. The vaccine according to claim 52, characterized in that, The oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated formulations, and ointments.

55. The vaccine according to claim 54, characterized in that, The tablets mentioned are sublingual tablets.

56. The vaccine according to claim 54, characterized in that, The granules are fine granules.

57. The vaccine according to claim 54, characterized in that, The powder is a granule.

58. The vaccine according to claim 54, characterized in that, The pills mentioned are small pills.

59. The vaccine according to any one of claims 44-51, characterized in that, The vaccine is in the form of a parenteral preparation.

60. The vaccine according to claim 59, characterized in that, The parenteral preparation is a transdermal preparation.

61. The vaccine according to claim 59, characterized in that, The parenteral preparation is an ointment or plaster.

62. The vaccine according to claim 59, characterized in that, The parenteral preparation is a topical liquid.

63. The vaccine according to claim 59, characterized in that, The parenteral preparation is an injectable preparation.

64. The vaccine according to claim 63, characterized in that, The injectable formulation is a push-in formulation.

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