Poxvirus recombinant chimeric antigen, immunogenic composition containing the same, and applications thereof

Abstract

The present invention relates to a recombinant chimeric antigen against poxvirus (especially monkeypox virus), an immunogenic composition containing the same, and their applications. The recombinant chimeric antigen of the present invention contains two immunogens: (1) the A35 protein of monkeypox virus or its antigenic fragment (or their derivative peptides) and (2) the M1 protein of monkeypox virus or its antigenic fragment (or their derivative peptides), which can stimulate an immune response against two infectious virus particles, namely intracellular mature virus particles (IMV) and extracellular enveloped virus particles (EEV), so as to efficiently stimulate a specific immune protection effect against monkeypox virus; in addition, the poxvirus vaccine of the present invention also has good safety, rapid responsiveness and production capacity support, and has excellent clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a poxvirus recombinant chimeric antigen, an immunogenic composition containing the same, and their applications. Background Art

[0002] Poxviruses, represented by monkeypox virus, are a type of large DNA viruses with a nuclear genome. The viral genome is about 130 - 375 kbp and can encode up to 200 viral proteins. In addition to encoding a large number of viral proteins, the viral particles of poxviruses are also relatively complex. They have two morphologically different infectious viral particles, called intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Among them, IMV has a single envelope and is more stable than EEV, mainly participating in the spread of the virus between hosts. While EEV has a special outer membrane structure and mainly participates in the dissemination of the virus within the host. Due to the different membrane structures, membrane components and cell infection mechanisms of IMV and EEV, their surface neutralizing antigens are also completely different.

[0003] Monkeypox is a viral zoonosis caused by infection with the monkeypox virus (MPXV). The monkeypox virus and smallpox virus belong to the genus Orthopoxvirus of the Poxviridae family, which includes four human pathogenic viruses, namely Variola virus (VARV), monkeypox virus, Cowpox virus (CPXV) and Vaccinia virus (VACV). The monkeypox virus was first isolated from laboratory monkeys by Danish scientists in 1958, and the first human infection case was found in the Democratic Republic of the Congo in 1970. After that, the monkeypox virus spread locally in central and western Africa for a long time, and evolved into two branches in West Africa and the Congo Basin (Central Africa). The Central African branch has strong transmission and pathogenicity, with a mortality rate of about 10.6%, and is mainly prevalent in African countries such as Gabon, Cameroon, Congo (Kinshasa), Congo (Brazzaville) and Sudan; the West African branch has weak transmission and pathogenicity, with a mortality rate of about 3.6%, and is mainly found in Nigeria, Liberia, Ghana and Sierra Leone and other western African countries and Europe and other non-African countries. Since the first case of monkeypox was confirmed in the UK in May 2022, confirmed cases of monkeypox have appeared in many countries including the United States, Italy, Sweden, Spain, Portugal, Belgium, Germany, and Australia, and the monkeypox virus is showing a global epidemic trend. On July 23, 2022, local time, the World Health Organization (WHO) officially announced that the monkeypox epidemic constituted a "public health emergency of international concern" (PHEIC), which is the highest level of alert issued by the WHO. As of October 7, 2022, a total of 71,237 cases of monkeypox have been reported worldwide, including 26 deaths, involving a total of 107 countries and regions. In addition, although the smallpox virus was eradicated in the 1980s, its pathogens still exist and may pose a threat to human health again.

[0004] Currently, there is no vaccine specifically developed for monkeypox virus in the world. There are only two vaccines that can be used to prevent monkeypox virus infection, namely JYNNEOS produced by Ankara-Bavarian Nordic and TM Vaccine (also known as Imvamune or Imvanex) and Sanofi Pasteur Both vaccines are attenuated live vaccines originally used to prevent smallpox; It is a second-generation vaccine that has the ability to replicate in the human body. After vaccination, there is a risk of encephalitis, myocarditis, progressive vaccinia infection, etc. It is not suitable for vaccination of young children, pregnant women and people with low or impaired immune function; JYNNEOS TMAs a third-generation vaccine, since it cannot replicate in the human body, its safety has been improved compared with the first-generation and second-generation vaccines, but the immune effect has also decreased to a certain extent. It should be noted that all of the above vaccine products were approved after the eradication of smallpox virus, so they have not been widely inoculated among the population, and their ability to control the transmission of smallpox virus and monkeypox virus and to eradicate the virus remains to be determined.

[0005] In short, all the currently approved vaccines are live attenuated vaccines developed against smallpox virus, and their ability to control the transmission of monkeypox virus remains to be verified. In addition, live attenuated viruses have obvious inoculation side effects and may face the risks and uncertainties of mutation leading to the re-strengthening of virus virulence. The above problems have limited the vaccinated population, so they are not suitable for infants, pregnant women, and people with low or impaired immune function.

[0006] In addition to the limited vaccinated population and obvious inoculation side effects, live attenuated vaccines also have uncertainties and potential safety hazards. One is the safety risk brought by the potential mutation of vaccine strains leading to the re-strengthening of virulence; the other is that poxvirus, as a large DNA virus with a nuclear quality, can encode up to 200 viral proteins. Therefore, the antigen components carried by live virus vaccines are extremely complex, and the effective immunogens and mechanisms of action are not clear. Among them, there are a variety of viral proteins with immunosuppressive functions, which will have a negative impact on the immunity of the vaccine and the immune system of the host. These safety hazards and uncertainties are particularly obvious in populations with low immunity, such as the elderly and HIV carriers, which further limit the vaccination of such immunocompromised populations.

[0007] Moreover, the live virus nature of live attenuated vaccines results in low production capacity and high cost, which cannot meet the needs of large-scale population vaccination and emergency vaccine production. Therefore, in view of the various drawbacks of existing vaccines, in response to the recent global epidemic trend of monkeypox virus and the possible future threats of poxviruses such as smallpox, it is urgent to develop a new generation of vaccines with clear immunogenic components, clear mechanisms of action, safety and effectiveness, and rapid availability with the help of new technologies to assist in disease prevention and control.

[0008] The information disclosed in this background art section is only intended to increase the understanding of the overall background of the present invention, and should not be regarded as an admission or any form of suggestion that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0009] Object of the Invention

[0010] In view of the various drawbacks of existing vaccines, the object of the present invention is to provide a recombinant chimeric antigen of monkeypox virus 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 recombinant chimeric antigen also have the advantages of safety, effectiveness, clear immunogenic components and protection mechanisms, high production capacity, low cost, etc., so as to meet the safety and production capacity requirements for large-scale vaccination of emergency vaccines.

[0011] Solution

[0012] To achieve the object of the present invention, the present invention provides the following technical solutions:

[0013] In a first aspect, the present invention provides a recombinant chimeric antigen of poxvirus, and the recombinant chimeric antigen comprises:

[0014] (1) Monkeypox virus A35 protein (encoded by the monkeypox virus A35R gene) or its antigenic fragment, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and having the same or substantially the same immunogenicity; and

[0015] (2) Monkeypox virus M1 protein (encoded by the monkeypox virus M1R gene) or its antigenic fragment, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and having the same or substantially the same immunogenicity.

[0016] In a feasible implementation, the antigenic fragment of the A35 protein is the extracellular segment of the protein or a part thereof; and / or, the antigenic fragment of the M1 protein is the extracellular segment of the protein or a part thereof.

[0017] In some embodiments, the recombinant chimeric antigen is in a single-chain form, and in each recombinant chimeric antigen chain, it comprises: more than 2 of the above-mentioned monkeypox virus A35 proteins or their antigenic fragments, and their amino acid sequences are the same or different; and, more than 1 of the above-mentioned monkeypox virus M1 proteins or their antigenic fragments.

[0018] Preferably, the recombinant chimeric antigen comprises 2 of the A35 proteins or their antigenic fragments and 1 of the M1 proteins or their antigenic fragments;

[0019] Further preferably, the recombinant chimeric antigen has a single-chain dimer structure. In a specific embodiment, the two A35 proteins or antigenic fragments thereof and one M1 protein or antigenic fragment thereof in the recombinant chimeric antigen are directly concatenated or concatenated through an appropriate linker sequence to form a single-chain polypeptide, and the obtained single-chain polypeptide can form a stable single-chain dimer structure under appropriate conditions (the two A35 proteins or antigenic fragments thereof dimerize to form a dimer).

[0020] In some specific embodiments, the recombinant chimeric antigen comprises an amino acid sequence arranged in the pattern of M-C1-A1-C2-A2, wherein:

[0021] M represents the monkeypox virus M1 protein or an antigenic fragment thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity as it,

[0022] A1 represents the monkeypox virus A35 protein or antigenic fragment I thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity as it,

[0023] A2 represents the monkeypox virus A35 protein or antigenic fragment II thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity as it,

[0024] C1 and C2 are each independently none, or a linker sequence (GGGGS)n, wherein n is any integer between 1 and 10; and,

[0025] wherein,

[0026] A1 and A2 are the same or different,

[0027] C1 and C2 are the same or different.

[0028] In some preferred specific embodiments, M represents the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence obtained by substituting, deleting or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO:1 and having the same or substantially the same immunogenicity as it;

[0029] and / or, A1 represents the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence obtained by substituting, deleting or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO:2 and having the same or substantially the same immunogenicity as it;

[0030] And / or, A2 represents the amino acid sequence shown in SEQ ID NO:2, or the amino acid sequence shown in SEQ ID NO:2 plus a fragment of 1-30 amino acids extended from its N-terminus to the A35 protein, or an amino acid sequence obtained by substituting, deleting or adding one or several amino acids to the above amino acid sequence and having the same or substantially the same immunogenicity as it; preferably, A2 represents the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence obtained by substituting, deleting or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO:3 and having the same or substantially the same immunogenicity as it.

[0031] More preferably, M represents the amino acid sequence shown in SEQ ID NO:1, A1 represents the amino acid sequence shown in SEQ ID NO:2, and A2 represents the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:3;

[0032] Preferably, both C1 and C2 are absent;

[0033] Even more preferably, the recombinant chimeric antigen comprises the amino acid sequence shown in SEQ ID NO:4.

[0034] Preferably, the N-terminus of the recombinant chimeric antigen further comprises a signal peptide sequence; optionally, the signal peptide sequence is as shown in SEQ ID NO:7;

[0035] Preferably, the C-terminus of the recombinant chimeric antigen further comprises a tag sequence; optionally, the tag is selected from at least one of Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, SUMO tag, and preferably His tag.

[0036] In the present invention, in order to balance the immune protection effects against both EEV and IMV virus particles, a recombinant chimeric antigen containing both the neutralizing antigen A35 of EEV and the neutralizing antigen M1 of IMV is designed. The two neutralizing antigens are encoded by the A35R gene and M1R gene of monkeypox virus respectively, and are homologous genes to vaccinia virus A33R and L1R.

[0037] In a second aspect, the present invention provides a method for preparing the recombinant chimeric antigen as described in the first aspect above, which comprises the following steps:

[0038] Add a Kozak sequence and the coding sequence of a signal peptide to the 5' end of the nucleotide sequence encoding the recombinant chimeric antigen as described in the first aspect above, and add the coding sequence of a histidine tag and a stop codon to the 3' end, then perform cloning and expression, screen for the correct recombinant, and then transfect the cells of the expression system for expression. Collect the cell culture supernatant and isolate the recombinant chimeric antigen therefrom.

[0039] In a feasible implementation manner of the above preparation method, the cells of the expression system are mammalian cells, insect cells, yeast cells or bacterial cells;

[0040] Optionally, the mammalian cells are HEK293T cells, 293F series cells or CHO cells; further optionally, the 293F series cells are HEK293F cells, Freestyle293F cells or Expi293F cells;

[0041] Optionally, the insect cells are sf9 cells, Hi5 cells, sf21 cells or S2 cells;

[0042] Optionally, the yeast cells are Pichia pastoris cells or yeast cells modified therefrom;

[0043] Optionally, the bacterial cells are Escherichia coli cells.

[0044] In the third aspect, the present invention provides a polynucleotide encoding the recombinant chimeric antigen as described in the first aspect above.

[0045] In a specific implementation manner, the polynucleotide is a nucleotide sequence optimized with human codons and can be DNA or mRNA;

[0046] Preferably, the polynucleotide is the DNA sequence shown in SEQ ID NO:5;

[0047] Preferably, the polynucleotide is the mRNA sequence shown in SEQ ID NO:6.

[0048] In the fourth aspect, the present invention provides a nucleic acid construct comprising the polynucleotide as described in the third aspect above, and optionally, at least one expression regulatory element operably linked to the polynucleotide.

[0049] In the fifth aspect, the present invention provides an expression vector comprising the nucleic acid construct as described in the fourth aspect above.

[0050] In the sixth aspect, the present invention provides a host cell transformed or transfected with the polynucleotide as described in the third aspect above, the nucleic acid construct as described in the fourth aspect above, or the expression vector as described in the fifth aspect above.

[0051] In a seventh aspect, the present invention provides the use of the recombinant chimeric antigen 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 preventing and / or treating poxvirus infection;

[0052] Preferably, the poxvirus is selected from: monkeypox virus, variola virus, vaccinia virus and / or cowpox virus;

[0053] Optionally, the medicament is a vaccine, preferably a recombinant protein vaccine; more preferably, the recombinant protein vaccine employs an adjuvant selected from the following: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant;

[0054] Optionally, the vaccine is in the form of a nasal spray, oral preparation, suppository or parenteral preparation;

[0055] Preferably, the nasal spray is selected from aerosols, sprays and powder sprays;

[0056] Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film coatings and ointments; more preferably, the tablet is a sublingual tablet; more preferably, the granule is a fine granule; more preferably, the powder is a powder; more preferably, the pill is a small pill;

[0057] Preferably, the parenteral preparation is a transdermal agent, ointment, plaster, topical liquid, injectable preparation; more preferably, the injectable preparation is a pushable preparation.

[0058] In an eighth aspect, the present invention provides a vaccine or immunogenic composition comprising the recombinant chimeric antigen 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, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

[0059] In some preferred specific embodiments, the vaccine or immunogenic composition is a monkeypox virus recombinant protein vaccine, which comprises the recombinant chimeric antigen as described in the first aspect above and an adjuvant;

[0060] Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant.

[0061] In some other preferred specific embodiments, the vaccine or immunogenic composition is a monkeypox virus DNA vaccine, which comprises:

[0062] (1) A eukaryotic expression vector; and

[0063] (2) A DNA sequence encoding the recombinant chimeric antigen as described in the first aspect above, preferably the DNA sequence shown in SEQ ID NO:5, which is constructed into the eukaryotic expression vector;

[0064] Optionally, the eukaryotic expression vector is selected from the pGX0001, pVAX1, pCAGGS, and pcDNA series vectors.

[0065] In some other preferred specific embodiments, the vaccine or immunogenic composition is a monkeypox virus mRNA vaccine, and the mRNA vaccine includes:

[0066] (I) An mRNA sequence encoding the recombinant chimeric antigen as described in the first aspect above, preferably the mRNA sequence shown in SEQ ID NO:6; and

[0067] (II) Lipid nanoparticles.

[0068] In some other preferred specific embodiments, the vaccine or immunogenic composition is a monkeypox virus - viral vector vaccine, which includes:

[0069] (1) A viral backbone vector; and

[0070] (2) A DNA sequence encoding the recombinant chimeric antigen as described in the first aspect above, preferably the DNA sequence shown in SEQ ID NO:5, which is constructed into the viral backbone vector;

[0071] Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, adeno - associated virus vector.

[0072] In a feasible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, oral preparation, suppository, or parenteral preparation;

[0073] Preferably, the nasal spray is selected from aerosols, sprays, and powder aerosols;

[0074] Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film - coated tablets, and pastes;

[0075] More preferably, the tablet is a sublingual tablet;

[0076] More preferably, the granule is a fine granule;

[0077] More preferably, the powder is a powder for external use;

[0078] More preferably, the pill is a small pill;

[0079] Preferably, the parenteral preparation is a transdermal agent, an ointment, a plaster, an external liquid preparation, an injectable preparation; More preferably, the injectable preparation is a pushable preparation.

[0080] 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 prophylactically and / or therapeutically effective amount of the following substances: the recombinant chimeric antigen 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, the host cell as described in the sixth aspect above, and / or the vaccine or immunogenic composition as described in the eighth aspect above.

[0081] The "prophylactically and / or therapeutically effective amount" may vary depending on the administration object, the target organ of the object, the symptoms, the administration method, etc., and can be determined according to the doctor's judgment by considering the type of dosage form, the administration method, the age and weight of the patient, the symptoms of the patient, etc.

[0082] Advantageous Effects

[0083] The inventors of the present invention designed a recombinant chimeric antigen against poxvirus (especially monkeypox virus), which contains two immunogens: (1) monkeypox virus A35 protein or its antigenic fragment (or their derivative peptide segments), and (2) monkeypox virus M1 protein or its antigenic fragment (or their derivative peptide segments); wherein, the former is a neutralizing antigen unique to intracellular mature virus particles (IMV), and the latter is a neutralizing antigen unique to extracellular enveloped virus particles (EEV); the vaccine containing both can stimulate an immune response against two infectious virus particles.

[0084] In a specific embodiment, the present invention directly concatenates two or more of the monkeypox virus A35 protein or its antigenic fragment with the monkeypox virus M1 protein or its antigenic fragment or concatenates them through an appropriate linking sequence, thereby forming a tandem fusion multivalent antigen in a single-chain form. In this tandem fusion multivalent antigen, A35 forms a stable dimer form, reversing the problem of the lack of immunogenicity of the A35 protein during single immunization; compared with the single immunization of the two proteins, the tandem fusion multivalent antigen not only retains the antigenicity of each of the two antigens, but also can more efficiently activate specific protective antibodies against monkeypox virus.

[0085] Compared with the poxvirus vaccines in the prior art, the vaccine products based on the recombinant chimeric antigen of the present invention have the following advantages:

[0086] 1) Subunit vaccines have better safety, thus overcoming the safety issues of existing live attenuated virus vaccines; at the same time, compared with live attenuated virus vaccines, subunit vaccines have the advantages of low production cost, rapid response, and production capacity support; verified by experiments, the poxvirus vaccine of the present invention has good efficacy;

[0087] 2) Using the antigenic sequences of the monkeypox virus itself, it has high specificity for the 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 poxviruses, there are still certain differences in their neutralizing antigen sequences and antigenic epitopes. Therefore, the protective effect against the monkeypox virus remains to be clarified. The poxvirus vaccine of the present invention is developed based on the antigenic epitopes of the monkeypox virus. Therefore, it has high specificity for the prevention and treatment of the monkeypox virus;

[0088] 3) There are a large number of proteins in the monkeypox virus, and the vast majority of them cannot stimulate effective antiviral immune responses, that is, they are ineffective components. In addition, there are some viral proteins with immunosuppressive effects; existing live virus vaccines cannot remove the above-mentioned ineffective and harmful components, so there are vaccination risks and uncertainties. The poxvirus vaccine of the present invention only retains 2 viral neutralizing antigens. Experimental data show that only these 2 antigens can exhibit complete protective effects in the mouse model. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] One or more embodiments are exemplarily illustrated by the pictures in the corresponding drawings. These exemplary illustrations do not constitute limitations on the embodiments. The special word "exemplary" here means "serving as an example, embodiment, or illustration". Any embodiment illustrated as "exemplary" here does not have to be construed as superior to or better than other embodiments.

[0090] Figure 1 Shows the gel filtration chromatography elution curves and SDS-PAGE identification results of the immunogenic proteins AA and MAA expressed in Example 2 of the present invention.

[0091] Figure 2 Shows the gel filtration chromatography elution curves and SDS-PAGE identification results of the single immunogenic proteins A35 and M1 expressed in Example 2 of the present invention.

[0092] Figure 3 Shows the affinity analysis results of the immunogenic proteins M1(A), MAA(B) binding to the neutralizing antibody 7D11 detected in Example 3 of the present invention.

[0093] Figure 4 Shows the affinity analysis results of the immunogenic proteins A35(A), AA(B), and MAA(C) binding to the neutralizing antibody A27D7 detected in Example 3 of the present invention.

[0094] Figure 5 It is a schematic diagram of the mouse immunization and challenge strategies adopted in Example 4 and Example 7 of the present invention.

[0095] Figure 6 It shows the specific binding antibody titers in the sera of immunized mice detected in Example 5 of the present invention, wherein, Figure 6 A is the specific antibody level against each immunogen, Figure 6 B is the specific antibody level against M1 and A35 epitopes.

[0096] Figure 7 It shows the neutralizing antibody titers of the sera of immunized mice against VACV live virus detected in Example 6 of the present invention.

[0097] Figure 8 It shows the pre-experiment results of infecting BALB / c mice by intranasal instillation with different doses of VACV-WR virus as described in Example 7 of the present invention, wherein, the abscissa shows the number of days after challenge, and the ordinate shows the survival percentage of the mice.

[0098] Figure 9 It shows the protective effects of each immunogenic protein against intranasal challenge of BALB / c mice with VACV-WR virus as described in Example 7 of the present invention, wherein, the abscissa shows the number of days after challenge, and the ordinate shows the percentage change in body weight (A) and the survival percentage (B) of the mice. Detailed implementation manners

[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 some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0100] In addition, to better illustrate the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In some embodiments, the raw materials, components, methods, means, etc. well-known to those skilled in the art are not described in detail so as to highlight the gist of the present invention.

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

[0102] Example 1: Immunogen Design

[0103] In this example, as a representative example of the present invention, two EEV neutralizing antigens A35 and one IMV neutralizing antigen M1R are concatenated in the arrangement of M1 - A35 - A35 from the N - terminus to the C - terminus. The formed single - chain fusion peptide can form a single - chain three - subunit structure with A35 - A35 dimerization, hereinafter referred to as MAA, which represents the recombinant chimeric antigen of the present invention.

[0104] The following sequences all adopt the sequence of the monkeypox virus isolate MPXV_USA_2022_MA001, and the GenBank number of the complete genome of this virus is ON563414.3.

[0105] In the MAA, the amino acid sequence of the M1 peptide segment is as shown in SEQ ID NO:1. Moreover, the two A35 neutralizing antigens adopt different A35 protein fragments. One is the S90 - T181 peptide segment of the A35 protein (its amino acid sequence is as shown in SEQ ID NO:2), and the other is the S64 - T181 peptide segment of the A35 protein (its amino acid sequence is as shown in SEQ ID NO:3). Thus, the amino acid sequence of the MAA is as shown in SEQ ID NO:4.

[0106] During the construction of the MAA, for the needs of protein expression and purification, the inventor also added a signal peptide sequence (as shown in SEQ ID NO:7) to the N - terminus of the MAA amino acid sequence shown in SEQ ID NO:4 and added 6 - histidine tags to its C - terminus, thereby forming the following complete construct shown in SEQ ID NO:8:

[0107] MAA Complete Construct (SEQ ID NO:8)

[0108]

[0109] (Among them, the underlined part at the front is the signal peptide sequence, the italic part is the amino acid sequence of the M1R peptide segment SEQ ID NO:1, the italic + underlined part is the amino acid sequence of the first A35 peptide segment SEQ ID NO:2, the bold part is the amino acid sequence of the second A35R peptide segment shown in SEQ ID NO:3, and the underlined part at the end is the histidine tag sequence).

[0110] In addition, for comparison, in this embodiment, an A35-A35 single-chain dimer structure formed only by tandemly connecting two EEV-neutralizing antigens A35R, hereinafter referred to as AA, was also designed. In the construction of AA, except for not containing the M1 peptide segment, the remaining sequences are the same as those of the above-mentioned MAA construction. Similarly, for the needs of protein expression and purification, the inventors also added a signal peptide sequence (as shown in SEQ ID NO:7) to its N-terminus and a 6-histidine tag to its C-terminus, thus forming the complete construct shown in SEQ ID NO:9 below:

[0111] AA Complete Construct (SEQ ID NO:9)

[0112]

[0113] (Among them, the underlined part at the front is the signal peptide sequence, the italic + underlined part is the amino acid sequence of the first A35R peptide segment, the bold part is the amino acid sequence of the second A35R peptide segment, and the underlined part at the end is the histidine tag sequence).

[0114] In addition, for comparison, this embodiment also designed an expression construct of the single antigen M1 and the A35 peptide segment. The amino acid sequences of the M1 and A35 peptide segments are as follows:

[0115] Amino acid sequence of the M1 peptide segment (SEQ ID NO:10):

[0116] MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAG;

[0117] Amino acid sequence of the A35 peptide segment (SEQ ID NO:11):

[0118] MSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT.

[0119] Example 2: Expression and purification of the immunogenic protein

[0120] Expression and Purification of Constructs MAA and AA

[0121] The amino acid sequences of the constructs MAA and AA designed in Example 1 were optimized for human codons to obtain nucleotide sequences encoding the MAA and AA antigens, as shown in SEQ ID NO:12 and SEQ ID NO:13 respectively; a Kozak sequence (GCCACC) was added to the 5' end of these nucleotide sequences, and a translation termination codon was added to the 3' end. Then, these DNA fragments were artificially synthesized and cloned between the EcoRI and XhoI restriction sites of the pCAGGS vector to obtain the expression plasmids of the constructs MAA and AA.

[0122] The above-mentioned expression plasmids of MAA and AA were transfected into 293F cells respectively for in vitro recombinant expression; 5 - 7 days after transfection, the cell supernatants were collected, which contained the expressed immunogenic proteins; then, protein purification was carried out; specifically, the cell supernatants containing the immunogenic proteins were roughly purified by nickel ion affinity chromatography (HisTrap TM HP (GE)), and then further purified by a gel filtration chromatography column Superdex200 10 / 300GL (GE). Finally, the protein purity and molecular weight were identified by SDS-PAGE.

[0123] The elution curve of gel filtration chromatography and the SDS-PAGE identification results are as Figure 1 shown, Figure 1 indicating that after two-step purification, highly pure AA and MAA proteins with molecular weights of ~25 kDa and ~50 kDa respectively can be obtained, meeting the expectations.

[0124] Expression and Purification of Single Immunogens M1 and A35

[0125] The amino acid sequences of the single antigens M1 and A35 peptide segments designed in Example 1 were optimized for human codons to obtain nucleotide sequences encoding M1 and A35 peptide segments, as shown in SEQ ID NO:14 and SEQ ID NO:15 respectively; a translation termination codon was added to the 3' end of these nucleotide sequences. Then, these DNA fragments were artificially synthesized and cloned into the pET-28a expression vector to obtain the expression plasmids of M1 and A35.

[0126] The expression plasmids of M1R and A35R were expressed through the Escherichia coli (E.coli) system, and the formed inclusion bodies were renatured in vitro into active proteins M1 and A35 by the method of arginine dilution renaturation; then, the renatured M1 or A35 protein was purified by a gel filtration chromatography column Superdex200 100 / 300GL. Finally, the protein purity and molecular weight were identified by SDS-PAGE.

[0127] The elution curve of gel filtration chromatography and the SDS-PAGE identification results are as Figure 2As shown, by Figure 2 it can be seen that after the above-mentioned gel filtration chromatography purification, high-purity M1 and A35 proteins can be obtained; among them, the elution position of M1 protein and SDS-PAGE both show ~20 kDa, which is in line with expectations; the elution position of A35 protein corresponds to a molecular weight of 25 kDa, while SDS-PAGE shows ~12 kDa. Thus, it can be known that A35 protein is a dimer formed by intermolecular interaction forces, which is in line with previous literature reports.

[0128] Example 3: Antigenicity Detection of Immunogen

[0129] In order to detect the exposure of each antigenic epitope of the recombinant chimeric antigen of the present invention and its antigenicity, in this example, by surface plasmon resonance (SPR) experimental method, the ability of the immunogen proteins (including single-chain fusion immunogen proteins MAA and AA and single antigen proteins M1 and A35) expressed and purified in Example 2 to bind neutralizing antibodies 7D11 and A27D7 was detected respectively; antibodies 7D11 and A27D7 are neutralizing antibodies against vaccinia virus antigens L1 and A33 respectively, and there is data indicating that they can cross-recognize M1 and A35 antigens of monkeypox virus. Among them, antibody A27D7 can recognize the dimeric epitope of A35.

[0130] The results are as Figure 3 and Figure 4 shown.

[0131] As can be seen from Figure 3 A and 3B, the affinity of single antigen protein M1 for neutralizing antibody 7D11 is 3.3 nM, showing a binding mode of slow binding and slow dissociation; while the recombinant chimeric antigen MAA of the present invention can be recognized by 7D11 but the affinity is slightly reduced, indicating that the M1 epitope on MAA is still exposed;

[0132] As can be seen from Figure 4 A-C, the affinity of single antigen protein A35 for antibody A27D7 is 2.2 μM, showing a binding mode of fast binding and fast dissociation; the affinity of single-chain fusion immunogen AA for antibody A27D7 is similar to that of single antigen A35; the affinity of the recombinant chimeric antigen MAA of the present invention for antibody A27D7 is similar to the above two, indicating that the A35 epitope on MAA is exposed and presents a stable dimeric form.

[0133] The above data all show that: the A35 and M1 antigenic epitopes on the recombinant chimeric antigen MAA of the present invention can be well exposed and have high antigenicity; in particular, the antigenicity of the A35 dimeric epitope therein has been greatly improved compared with single antigen protein A35 or its dimer AA.

[0134] Example 4: Mouse Immunization Experiment

[0135] To verify the immune protection efficacy of the recombinant chimeric antigen of the present invention, the inventors separately mixed and emulsified each immunogenic protein obtained in Example 2 with AddaVax TM adjuvant, and immunized mice according to the strategy of Figure 5 . The replicable vaccinia virus Tian Tan strain (VACV-VTT) was used as the live attenuated vaccine control group.

[0136] In the immunization experiment, all mice used were female BALB / c mice, 6-8 weeks old, with an average weight of 15-20 g. Each experimental group used 6 mice. As Figure 4 shown, the mice were immunized on days 0, 21, and 42 respectively, for a total of 3 times, with a dose of 10 μg / mouse each time; the inoculation method was intramuscular injection, and the injection site was on the thigh of the mouse, with 50 μL injected into each of the left and right legs.

[0137] VACV-VTT was used to immunize mice by scratching the tail root. Specifically, the immunization was carried out on day 0, for a total of one immunization, with an immunization dose of 10 7 PFU / mouse.

[0138] On the two days before the second immunization, the third immunization, and the challenge (i.e., days 19, 40, and 54), blood was collected from the eyes of all groups of mice. After standing for coagulation and centrifuging at 1500 rpm for 10 minutes, mouse serum was obtained, and the mouse serum was immediately aliquoted and stored in a -80°C refrigerator for subsequent ELISA detection of specific antibody titers and determination of live virus neutralization ability.

[0139] Example 5: ELISA experiment to detect the specific antibody titer induced by the vaccine

[0140] Each immunogenic protein used for mouse immunization (i.e., single antigen proteins A35R and M1R, single-chain fusion immunogens AA and MAA, prepared in Example 2) was diluted to 2 μg / mL with ELISA coating buffer (Solarbio, C1050). 100 μL of the above-diluted immunogenic protein or cell lysate after infection with Vaccinia virus Tian Tan strain (for detecting the antibody titer of the live attenuated vaccine immunization group) was added to each well of a 96-well ELISA plate (Corning, 3590), and it was left standing overnight at 4°C. The coating buffer was discarded, and PBS was added to wash away the residual coating buffer. Then, 100 μL of ELISA blocking buffer (10% skim milk powder prepared with PBST) was added and left standing at room temperature for 1 hour for blocking. During the blocking period, the serum of immunized mice was diluted with ELISA blocking buffer, starting from 200-fold and performing 3-fold serial dilutions. 11 dilution factors were made for each sample. After the blocking was completed, the blocking buffer was removed, and the serum of immunized mice diluted 10-fold with the blocking buffer was added to the ELISA plate, 100 μL for each dilution factor, and incubated at room temperature for 1 hour. Then, it was washed 3 times with PBST. Subsequently, HRP-labeled goat anti-mouse secondary antibody (Abcam, ab6789) diluted 1:4000 with the blocking buffer was added, incubated at room temperature for 1 hour, then washed 5 - 6 times with PBST, and TMB chromogenic solution was added for color development. After reacting for an appropriate time, 2M hydrochloric acid was added to terminate the reaction. The OD450 reading was detected on an ELISA reader. A sample with an OD450 value greater than 2.5 times that of the negative control was determined to be positive, and the highest dilution factor of the serum determined to be positive was defined as the serum antibody titer (Endpoint titer). When the reaction value of the lowest dilution factor was still less than 2.5 times that of the negative control, the titer of this sample was defined as half of the lowest dilution factor, i.e., Log10 = 1.

[0141] The detection results of the serum specific antibody titers of the mice immunized with each immunogenic protein are as Figure 6 shown, where Figure 6 A is the specific antibody level against each immunogen, Figure 6 B is the specific antibody level against the M1 and A35 epitopes; as can be seen from Figure 6 A: The single-chain fusion antigen MAA of the present invention can well stimulate specific antibodies after the first immunization, and moreover, the specific antibody titers after each immunization are superior to all control groups including the live attenuated vaccine immunization group. It is worth mentioning that for the A35 epitope, immunization with the A35 protein alone cannot stimulate specific antibodies, indicating a lack of immunogenicity, while the single-chain fusion-modified AA and MAA can effectively stimulate specific antibody responses against the A35 epitope and have good immunogenicity ( Figure 6 B); furthermore, MAA can simultaneously stimulate specific antibody responses against the A35 and M1 epitopes at a high level, among which the ability to stimulate A35 epitope antibodies is superior to that of the modified AA alone immunization, and the ability to stimulate M1 epitope antibodies is similar to that of M1 alone immunization.Figure 6 B);

[0142] Example 6: Determination of the neutralizing ability of immune serum against live VACV

[0143] Vaccinia virus (VACV) is the prototype virus of the genus Orthopoxvirus. Due to the high homology among the major immunogens of the genus Orthopoxvirus, VACV with a lower biosafety level and easy to operate is commonly used internationally for the evaluation of the neutralizing ability at the cellular level of other orthopoxvirus vaccines and the evaluation of the protective efficacy in mouse models. Therefore, in this example, the mouse-adapted strain Western Reserve of VACV (VACV-WR), which is commonly used internationally, was used to determine the neutralizing ability of live virus at the cellular level of each immunogenic protein.

[0144] The immune mouse sera obtained in Example 4 were diluted with a medium (DMEM) containing 2% inactivated serum, and two-fold serial dilutions were performed starting from 20-fold, with 10 dilution degrees for each sample. The VACV-WR virus was also diluted to 500 PFU / mL with the same diluent. 200 μL of the diluted immune mouse sera (the diluent without immune mouse sera was used as the 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 a 12-well plate one day in advance, and the optimal density was about 95% on the next day. The medium in the 12-well plate was discarded, and after washing away the residual medium with PBS, the incubated serum-virus mixture was added to the 12-well plate and infected at 37 °C for 2 hours. After infection, the virus-serum mixture was removed, the residual virus was washed away with PBS, and the prepared carboxymethylcellulose-DMEM mixture (a 1:1 mixture of 2% carboxymethylcellulose and 2×DMEM) was added and cultured at 37 °C for 48 - 60 hours. After obvious CPE was observed under the microscope, 4% paraformaldehyde fixative was added and fixed at room temperature for 2 hours, followed by crystal violet staining and counting.

[0145] PRNT 50 The calculation method is as follows: The wells containing only the virus were used as the control wells. The number of plaques in all experimental wells after counting was divided by the number of plaques in the control wells, which was the inhibition rate of the serum in each experimental well against the virus. Then, PRNT was calculated using the log(inhibitor) vs. normalized response--Variable slope formula in GraphPad software. 50 .

[0146] The results are shown in Figure 7 .

[0147] Since the VACV-WR strain used in the live virus neutralization experiment hardly produces EEV, all our neutralization experiments used IMV.

[0148] As Figure 7 shown, since A35 does not exist on the IMV virions, the A35 and AA immunized groups cannot exhibit neutralizing ability in terms of the neutralization level of live virus; while the M1, MAA and VACV-VTT immunized groups can all produce neutralizing antibodies against the IMV virions. Among them, the ability to stimulate neutralizing antibodies of MAA is significantly higher than that of M1 and the attenuated live vaccine VACV-VTT immunized group.

[0149] Example 7: Viral challenge protection experiment

[0150] When the VACV-WR virus strain is intranasally infected into BALB / c mice, it can cause the death of the mice. To determine the appropriate challenge dose, we first conducted a pre-experiment of intranasal infection of BALB / c mice at 17-19 weeks of age (the same age as that at the time of challenge after three immunizations) with different doses of VACV-WR virus to measure the LD50 of VACV-WR infecting BALB / c mice in our experimental system. The results of the pre-experiment are as Figure 8 shown. When the challenge dose is 2×10 5 PFU, all the mice died within 7 days after infection. By calculation, the virus amount at this time is 7LD50, and we subsequently used this virus amount as the challenge dose for the animal experiment.

[0151] As Figure 5 shown, the mice immunized three times in Example 4 were subjected to a VACV-WR virus intranasal challenge experiment with 7LD50 two weeks after the third immunization, that is, on the 56th day, to evaluate the protective effect of the vaccine in the animal model. The results of the percentage change in body weight and the survival percentage of the mice in each challenge experimental group are shown in Figure 9 A and Figure 9 B.

[0152] Figure 9 The results of A and 9B show that the single antigen A35 immunized group has no protective effect on the mice infected with the VACV-WR virus, and all the mice died within 6 days after the challenge, which is consistent with the result that it cannot effectively stimulate specific antibodies; although the single antigen M1 and the single-chain fusion immunogen AA showed a relatively high level of specific antibody in Example 4 and a relatively high level of ability to stimulate neutralizing antibodies in Example 5, they can only protect 60% of the mice from death caused by VACV-WR infection, and the surviving mice also showed a significant decrease in body weight; compared with the above groups, the single-chain fusion immunogen MAA of the present invention can provide 100% protection for the mice infected with VACV-WR, and there is no obvious change in body weight.

[0153] The above results indicate that: the single-chain fusion immunogen MAA of the present invention shows excellent protective effect in the mouse model.

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

[0155] Sequences Involved in This Application:

[0156] SEQ ID NO:1 (Amino acid sequence of M1 in MAA)

[0157]

[0158] SEQ ID NO:2 (Amino acid sequence of the first A35 in MAA)

[0159]

[0160] SEQ ID NO:3 (Amino acid sequence of the second A35 in MAA)

[0161]

[0162] SEQ ID NO:4 (Amino acid sequence of MAA)

[0163]

[0164] SEQ ID NO:5 (DNA sequence encoding MAA as shown in SEQ ID NO:4)

[0165] GCCGCTGCCTCTATCCAAACAACCGTGAATACCCTGAGCGAGAGAATCTCCTCCAAGCTTGAGC

[0166] AGGAGGCCAACGCCAGCGCTCAGACAAAGTGCGACATCGAGATCGGCAACTTCTACATCAGAC

[0167] AGAACCACGGCTGCAATATCACCGTGAAGAACATGTGCAGCGCTGACGCCGACGCCCAGCTGG

[0168] ACGCCGTGCTGTCTGCCGCTACCGAAACCTACAGCGGCCTGACACCTGAGCAGAAAGCCTACG

[0169] TTCCTGCCATGTTCACCGCCGCCCTGAACATTCAGACAAGCGTGAACACCGTGGTGCGGGACT

[0170] TCGAGAACTACGTGAAGCAGACCTGTAACAGCAGCGCCGTGGTGGACAACAAGCTGAAGATC

[0171] CAGAATGTGATCATCGACGAGTGCTACGGCGCTCCCGGCAGCCCTACCAACCTGGAGTTCATCA

[0172] ACACAGGCTCTAGCAAGGGCAATTGCGCTATCAAGGCCCTGATGCAGCTGACCACAAAAGCCA

[0173] CAACCCAAATTGCCCCAAGACAGGTGGCTGGCTCTACCACACAGTACGATCACAAGGAAAGCT

[0174] GTAACGGCCTGTATTACCAGGGCAGCTGCTACATCCTGCATTCTGACTACAAGTCATTTGAGGAT

[0175] GCCAAGGCCAACTGCGCCGCCGAGAGCAGCACCCTGCCTAACAAGTCCGACGTGCTGACCAC

[0176] CTGGCTGATCGACTACGTGGAAGATACCTGGGGAAGCGATGGCAACCCCATCACCAAGACCAC

[0177] ATCCGACTATCAGGATAGCGACGTGTCTCAGGAGGTGCGCAAGTACTTTTGTACCAGCGCAAAT

[0178] AAAGCCGCCATCACAGACAGCGCTGTCGCCGTGGCCGCCGCCAGCTCAACACACAGAAAAGT

[0179] GGTGTCCAGTACGACCCAGTACGACCACAAGGAATCTTGTAATGGCCTGTACTACCAGGGATCT

[0180] TGCTACATCCTGCACAGCGACTACAAGAGCTTCGAGGATGCCAAGGCCAACTGCGCCGCCGAA

[0181] AGCAGCACCCTGCCCAACAAGAGCGATGTGCTGACAACCTGGCTCATCGACTATGTCGAGGAC

[0182] ACCTGGGGCAGCGACGGTAACCCTATCACCAAAACCACAAGCGATTACCAGGACTCTGATGTG

[0183] TCCCAAGAAGTGCGGAAGTACTTCTGCACC

[0184] SEQ ID NO:6 (mRNA sequence encoding MAA as shown in SEQ ID NO:4)

[0185] GCCGCUGCCUCUAUCCAAACAACCGUGAAUACCCUGAGCGAGAGAAUCUCCUCCAAGCUUG

[0186] AGCAGGAGGCCAACGCCAGCGCUCAGACAAAGUGCGACAUCGAGAUCGGCAACUUCUACAU

[0187] CAGACAGAACCACGGCUGCAAUAUCACCGUGAAGAACAUGUGCAGCGCUGACGCCGACGCC

[0188] CAGCUGGACGCCGUGCUGUCUGCCGCUACCGAAACCUACAGCGGCCUGACACCUGAGCAGA

[0189] AAGCCUACGUUCCUGCCAUGUUCACCGCCGCCCUGAACAUUCAGACAAGCGUGAACACCGU

[0190] GGUGCGGGACUUCGAGAACUACGUGAAGCAGACCUGUAACAGCAGCGCCGUGGUGGACAA

[0191] CAAGCUGAAGAUCCAGAAUGUGAUCAUCGACGAGUGCUACGGCGCUCCCGGCAGCCCUACC

[0192] AACCUGGAGUUCAUCAACACAGGCUCUAGCAAGGGCAAUUGCGCUAUCAAGGCCCUGAUGC

[0193] AGCUGACCACAAAAGCCACAACCCAAAUUGCCCCAAGACAGGUGGCUGGCUCUACCACACA

[0194] GUACGAUCACAAGGAAAGCUGUAACGGCCUGUAUUACCAGGGCAGCUGCUACAUCCUGCAU

[0195] UCUGACUACAAGUCAUUUGAGGAUGCCAAGGCCAACUGCGCCGCCGAGAGCAGCACCCUGC

[0196] CUAACAAGUCCGACGUGCUGACCACCUGGCUGAUCGACUACGUGGAAGAUACCUGGGGAAG

[0197] CGAUGGCAACCCCAUCACCAAGACCACAUCCGACUAUCAGGAUAGCGACGUGUCUCAGGAG

[0198] GUGCGCAAGUACUUUUGUACCAGCGCAAAUAAAGCCGCCAUCACAGACAGCGCUGUCGCCG

[0199] UGGCCGCCGCCAGCUCAACACACAGAAAAGUGGUGUCCAGUACGACCCAGUACGACCACAA

[0200] GGAAUCUUGUAAUGGCCUGUACUACCAGGGAUCUUGCUACAUCCUGCACAGCGACUACAAG

[0201] AGCUUCGAGGAUGCCAAGGCCAACUGCGCCGCCGAAAGCAGCACCCUGCCCAACAAGAGCG

[0202] AUGUGCUGACAACCUGGCUCAUCGACUAUGUCGAGGACACCUGGGGCAGCGACGGUAACCC

[0203] UAUCACCAAAACCACAAGCGAUUACCAGGACUCUGAUGUGUCCCAAGAAGUGCGGAAGUAC

[0204] UUCUGCACC

[0205] SEQ ID NO:7 (Amino acid sequence of the signal peptide)

[0206]

[0207] SEQ ID NO:8 (Amino acid sequence of the complete MAA construct in Example 1)

[0208]

[0209] SEQ ID NO:9 (Amino acid sequence of the complete AA construct in Example 1)

[0210]

[0211] SEQ ID NO:10 (Amino acid sequence of the single antigen M1 peptide in Example 1)

[0212] MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCS

[0213] ADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQT

[0214] CNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQI

[0215] APRQVAG

[0216] SEQ ID NO:11 (Amino acid sequence of the single antigen A35 peptide in Example 1)

[0217] MSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLI

[0218] DYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT

[0219] SEQ ID NO:12 (Nucleotide sequence encoding the complete MAA construct)

[0220]

[0221] SEQ ID NO:13 (Nucleotide sequence encoding the complete AA construct)

[0222]

[0223]

[0224] SEQ ID NO:14 (Nucleotide sequence encoding the single antigen M1 peptide (i.e., SEQ ID NO:10))

[0225] atgggagcagctgcgtcaatacaaacaactgtaaacaccctgagcgaacgtattagctccaaacttgagcaagaggcaaacgcgagcgcgcaaacgaaatgcgatattgagatcggcaacttctatatccgccaaaatcacggttgtaatattaccgtcaagaacatgtgcagcgcggacgcggacgcgcagctggacgccgttttgtctgcagcgaccgaaacctattccggtctgaccccggagcagaaagcgtacgttccggctatgttcaccgcagcactcaatatccaaaccagcgtcaataccgttgttcgtgattttgaaaattacgtgaagcagacgtgcaactcctcggcggtggtggataacaaactgaagatccaaaacgtgattatcgacgaatgttacggcgctccgggttctccgaccaacttggagtttatcaacactggcagcagcaaaggcaactgcgctattaaggcgctgatgcagctgactacaaaggcgaccacgcagatcgccccacgtcaggtggccggt

[0226] SEQ ID NO:15 (Nucleotide sequence encoding the single antigen A35 peptide (i.e., SEQ ID NO:11))

[0227] atgtcaactacacaatatgatcacaaagaaagttgcaacggcttatattatcagggttcttgttatatcctgcacagcgactacaagtcttttgaggatgcgaaagctaactgcgcggcagaaagcagcaccctgccgaataagtccgacgtgttgaccacgtggctgatcgattacgtggaagatacctggggttccgacggcaacccgattaccaaaactacgagcgactaccaggatagcgacgtttcgcaagaggttcgtaaatacttctgcacc

Claims

1. A poxvirus recombinant chimeric antigen, characterized in that, The amino acid sequence of the poxvirus recombinant chimeric antigen is an amino acid sequence arranged in the M-C1-A1-C2-A2 pattern, where: M represents the antigenic fragment of the monkeypox virus M1 protein, and its amino acid sequence is as shown in SEQ ID NO:1; A1 represents the antigenic fragment I of the monkeypox virus A35 protein, and its amino acid sequence is as shown in SEQ ID NO:2; A2 represents the antigenic fragment II of the monkeypox virus A35 protein, and its amino acid sequence is as shown in SEQ ID NO:3; C1 and C2 are each independently none or a linker sequence (GGGGS)n, where n is any integer between 1 and 10.

2. The recombinant chimeric antigen of vaccinia virus according to claim 1, characterized in that, The amino acid sequence of the poxvirus recombinant chimeric antigen is as shown in SEQ ID NO:

4.

3. A poxvirus recombinant fusion antigen, characterized in that, The fusion antigen is composed of the following sequences in the order from the N-terminus to the C-terminus: a signal peptide sequence, the amino acid sequence of the poxvirus recombinant chimeric antigen as described in any one of claims 1-2, and a tag sequence; Wherein, the tag is one selected from the group consisting of Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, SUMO tag.

4. The fusion antigen according to claim 3, wherein The signal peptide sequence is as shown in SEQ ID NO:7; And / or, the tag is His tag.

5. The fusion antigen according to claim 3, wherein The amino acid sequence of the fusion antigen is as shown in SEQ IDNO:

8.

6. A method for preparing the poxvirus recombinant chimeric antigen according to any one of claims 1-2, comprising the following steps: Add a Kozak sequence and the coding sequence of the signal peptide to the 5' end of the nucleotide sequence encoding the poxvirus recombinant chimeric antigen as described in any one of claims 1-2, add the coding sequence of the histidine tag and a stop codon to the 3' end, perform cloning and expression, screen the correct recombinant, and then transfect the expression system cells for expression, collect the cell culture supernatant, and isolate the poxvirus recombinant chimeric antigen therefrom.

7. The preparation method according to claim 6, characterized in that: The expression system cells are mammalian cells, yeast cells or bacterial cells.

8. The preparation method according to claim 7, characterized in that: The mammalian cells are HEK293T cells, 293F series cells or CHO cells; And / or, the yeast cells are Pichia pastoris cells or yeast cells modified therefrom; And / or, the bacterial cells are Escherichia coli cells.

9. The preparation method according to claim 8, wherein, The 293F series cells are HEK293F cells, Freestyle293F cells or Expi293F cells.

10. A polynucleotide encoding the poxvirus recombinant chimeric antigen as described in any one of claims 1-2 or the fusion antigen as described in any one of claims 3-5.

11. The polynucleotide according to claim 10, wherein: The polynucleotide is DNA or mRNA.

12. The polynucleotide according to claim 11, wherein: The polynucleotide contains the DNA sequence as shown in SEQ ID NO:

5.

13. The polynucleotide according to claim 11, wherein: The polynucleotide contains the mRNA sequence as shown in SEQ ID NO:

6.

14. A nucleic acid construct comprising the polynucleotide as described in any one of claims 10-13, and at least one expression regulatory element operably linked to the polynucleotide.

15. An expression vector comprising the nucleic acid construct as described in claim 14.

16. A host cell, which is transformed or transfected with the polynucleotide according to any one of claims 10-13, the nucleic acid construct according to claim 14, or the expression vector according to claim 15.

17. Use of the recombinant chimeric poxvirus antigen according to any one of claims 1-2, the fusion antigen according to any one of claims 3-5, the polynucleotide according to any one of claims 10-13, the nucleic acid construct according to claim 14, the expression vector according to claim 15, or the host cell according to claim 16 in the preparation of a vaccine for preventing poxvirus infection; Among them, The poxvirus is selected from: monkeypox virus and / or vaccinia virus.

18. A vaccine or immunogenic composition, which comprises the recombinant chimeric poxvirus antigen according to any one of claims 1-2, the fusion antigen according to any one of claims 3-5, the polynucleotide according to any one of claims 10-13, the nucleic acid construct according to claim 14, the expression vector according to claim 15, or the host cell according to claim 16, and a physiologically acceptable vehicle, adjuvant, excipient, carrier, and / or diluent.

19. The vaccine or immunogenic composition according to claim 18, which is a recombinant protein vaccine against monkeypox virus and comprises: The recombinant chimeric poxvirus antigen according to any one of claims 1-2 or the fusion antigen according to any one of claims 3-5, and an adjuvant.

20. The vaccine or immunogenic composition according to claim 19, wherein: The adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, and MF59-like adjuvant.

21. The vaccine or immunogenic composition according to claim 18, which is a monkeypox virus DNA vaccine, and the DNA vaccine comprises: (1) A eukaryotic expression vector; and (2) A DNA sequence encoding the recombinant chimeric poxvirus antigen according to any one of claims 1-2 or the fusion antigen according to any one of claims 3-5, which is constructed into the eukaryotic expression vector.

22. The vaccine or immunogenic composition according to claim 21, wherein: The DNA sequence is the DNA sequence shown in SEQ ID NO:5; and / or, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS, and pcDNA series vectors.

23. The vaccine or immunogenic composition according to claim 18, which is a monkeypox virus mRNA vaccine, and the mRNA vaccine comprises: (I) An mRNA sequence encoding the recombinant chimeric poxvirus antigen according to any one of claims 1-2 or the fusion antigen according to any one of claims 3-5; and (II) A lipid nanoparticle.

24. The vaccine or immunogenic composition according to claim 23, characterized in that: The mRNA sequence is the mRNA sequence shown in SEQ ID NO:

6.

25. The vaccine or immunogenic composition according to claim 18, which is a monkeypox virus - viral vector vaccine, and it comprises: (1) A viral backbone vector; and (2) A DNA sequence encoding the recombinant chimeric poxvirus antigen according to any one of claims 1-2 or the fusion antigen according to any one of claims 3-5, which is constructed into the viral backbone vector.

26. The vaccine or immunogenic composition according to claim 25, characterized in that: The DNA sequence is the DNA sequence shown in SEQ ID NO:5; And / or, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector.

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