Poxvirus recombinant chimeric antigen, its subunit vaccine and its application

Abstract

The present invention relates to a recombinant chimeric antigen targeting poxvirus (particularly monkeypox virus), a subunit vaccine thereof, and its application. The recombinant chimeric antigen of the present invention comprises two immunogens arranged in a specific manner: monkeypox virus A35 protein or its antigenic fragment (or its derivative peptide segment) and monkeypox virus M1 protein or its antigenic fragment (or its derivative peptide segment), which can stimulate immune responses against two infectious virus particles: intracellular mature virus particles (IMV) and extracellular enveloped virus particles (EEV), thereby efficiently stimulating specific immune protection 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 specifically relates to a poxvirus recombinant chimeric antigen, a subunit vaccine thereof and applications thereof. Background Art

[0002] Poxviruses, represented by monkeypox virus, are a type of large nucleocytoplasmic DNA virus with a viral genome of approximately 130-375kbp, capable of encoding up to 200 viral proteins. In addition to the complex encoding of viral proteins, the viral particles of poxviruses are also relatively complex. They have two morphologically different infectious viral particles, called intracellular mature viruses (IMVs) and extracellular enveloped viruses (EEVs). Among them, IMV has a layer of envelope, which is more stable than EEV and is mainly involved in the spread of the virus between hosts. EEV, on the other hand, has a special outer membrane structure, which is mainly involved in the spread of the virus in the host. Because IMV and EEV have different membrane structures, membrane components and cell infection mechanisms, their surface neutralizing antigens are also completely different.

[0003] Monkeypox is a viral zoonosis caused by infection with the monkeypox virus (MPXV). Monkeypox virus and smallpox virus belong to the same family, the Poxviridae, and the genus Orthopoxvirus. This genus includes four human pathogenic viruses: variola virus (VARV), monkeypox virus, cowpox virus (CPXV), and vaccinia virus (VACV). Monkeypox virus was first isolated from laboratory monkeys by Danish scientists in 1958, and the first human case was discovered in the Democratic Republic of the Congo in 1970. Since then, monkeypox virus has long been locally transmitted in west-central Africa, evolving into two branches: West Africa and the Congo Basin (Central Africa). The Central African lineage has a high transmission capacity and pathogenicity, with a case fatality rate of approximately 10.6%. It is predominantly found in African countries such as Gabon, Cameroon, the Democratic Republic of the Congo, the Republic of the Congo, and Sudan. The West African lineage has a lower transmission capacity and pathogenicity, with a case fatality rate of approximately 3.6%. It is primarily found in West African countries such as Nigeria, Liberia, Ghana, and Sierra Leone, as well as in Europe and other non-African countries. Since the first case of monkeypox was confirmed in the United Kingdom in May 2022, confirmed cases have been reported in several countries, including the United States, Italy, Sweden, Spain, Portugal, Belgium, Germany, and Australia, demonstrating the global prevalence of the monkeypox virus. On July 23, 2022, the World Health Organization (WHO) officially declared the monkeypox outbreak a Public Health Emergency of International Concern (PHEIC), the highest level of alert issued by the WHO following the 2020 COVID-19 pandemic. As of October 7, 2022, a total of 71,237 cases of monkeypox, including 26 deaths, have been reported worldwide, affecting 107 countries and regions. In addition, although the smallpox virus was eradicated in the 1980s, its pathogens still exist and may threaten 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: 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. It carries the risk of encephalitis, myocarditis, and progressive vaccinia infection after vaccination. It is also not suitable for young children, pregnant women, and people with low or compromised immune function. TMThese are third-generation vaccines. Because they cannot replicate in the human body, they offer improved safety compared to first- and second-generation vaccines, but their immune effectiveness is also somewhat reduced. It's worth noting that these vaccines were approved after the eradication of smallpox virus, and therefore haven't been widely administered to the general population. Their effectiveness in curbing the spread of smallpox and monkeypox viruses and eradicating them remains to be determined.

[0005] In short, all currently approved vaccines are attenuated live vaccines developed for smallpox virus, and their ability to curb the spread of monkeypox virus remains to be verified. Furthermore, attenuated live viruses carry significant side effects and carry the risk and uncertainty of mutations that could lead to a reversion to increased virulence. These issues limit the vaccine's intended recipient population, making it unsuitable for young children, pregnant women, and those with weakened or compromised immune systems.

[0006] In addition to limited vaccine populations and clear side effects, attenuated live vaccines also present uncertainties and safety risks. First, there is the safety risk of potential vaccine strain mutations leading to a reversion to virulence. Second, poxviruses are large, nucleocytoplasmic DNA viruses whose viral genomes can encode up to 200 viral proteins. Therefore, the antigenic composition of live virus vaccines is extremely complex, with unclear effective immunogens and mechanisms of action. These vaccines include multiple viral proteins with immunosuppressive properties, which can negatively impact both the vaccine's effectiveness and the host's immune system. These safety risks and uncertainties are particularly pronounced in immunocompromised populations, such as the elderly and HIV carriers, further limiting vaccination of these vulnerable populations.

[0007] Furthermore, the live virus nature of MLV vaccines results in low production capacity and high costs, making them unable to meet the needs of large-scale vaccination and emergency vaccine production. Therefore, given the various drawbacks of existing vaccines, the recent global epidemic of monkeypox virus and the potential future threat posed by poxviruses such as smallpox, 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, effectiveness, and rapid availability to aid disease prevention and control.

[0008] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person 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 the present invention is to provide a recombinant chimeric antigen of monkeypox virus that can efficiently stimulate a specific immune response against monkeypox virus (for example, producing protective antibodies), its related vaccine products, its preparation method and application; in addition, vaccine products based on this recombinant chimeric antigen also have the advantages of safety, effectiveness, clear immunogenic composition and protection mechanism, high production capacity, low cost, etc., thereby meeting the safety and production capacity requirements of large-scale vaccination of emergency vaccine populations.

[0011] Solution

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

[0013] In a first aspect, the present invention provides a poxvirus recombinant chimeric antigen, wherein the recombinant chimeric antigen comprises an amino acid sequence arranged according to the following formula (I):

[0014] A1-C1-M1-C2-A2-C3-M2

[0015] (I)

[0016] In formula (I):

[0017] A1 represents the monkeypox virus A35 protein (encoded by the monkeypox virus A35R gene) or antigenic fragment I thereof, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity thereto,

[0018] A2 represents the monkeypox virus A35 protein (encoded by the monkeypox virus A35R gene) or antigenic fragment II thereof, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity thereto,

[0019] M1 represents the monkeypox virus M1 (encoded by the monkeypox virus M1R gene) protein or antigenic fragment I thereof, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity thereto,

[0020] M2 represents the monkeypox virus M1 protein (encoded by the monkeypox virus M1R gene) or antigenic fragment II thereof, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity thereto;

[0021] C1, C2, and C3 are each independently none or a linker sequence (GGGGS)n, wherein n is any integer between 1 and 10;

[0022] in,

[0023] A1 and A2 are the same or different,

[0024] M1 and M2 are the same or different.

[0025] In a feasible implementation, the antigenic fragment I or II of the A35 protein is the extracellular segment of the protein or a part thereof; and / or, the antigenic fragment I or II of the M1 protein is the extracellular segment of the protein or a part thereof.

[0026] In some embodiments:

[0027] A1 represents the amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO: 1, and having the same or substantially the same immunogenicity as the amino acid sequence;

[0028] And / or, A2 represents the amino acid sequence as shown in SEQ ID NO: 1, or the amino acid sequence of the amino acid sequence as shown in SEQ ID NO: 1 plus the amino acid sequence of a fragment extending 1-30 amino acids therefrom to the N-terminus of the A35 protein, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids from the above amino acid sequence, and having the same or substantially the same immunogenicity as the above amino acid sequence; preferably, A2 represents the amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids from the amino acid sequence as shown in SEQ ID NO: 2, and having the same or substantially the same immunogenicity as the above amino acid sequence;

[0029] And / or, M1 is the same as M2, representing the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 3 and having the same or substantially the same immunogenicity.

[0030] In a preferred embodiment:

[0031] Said A1 represents the amino acid sequence shown in SEQ ID NO: 1;

[0032] A2 represents the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0033] The M1 is identical to M2 and represents the amino acid sequence shown in SEQ ID NO: 3.

[0034] Further preferably, said A1 represents the amino acid sequence shown in SEQ ID NO: 1;

[0035] A2 represents the amino acid sequence shown in SEQ ID NO: 2;

[0036] The M1 is identical to M2 and represents the amino acid sequence shown in SEQ ID NO: 3.

[0037] Optionally, C1, C2, and C3 are all absent.

[0038] More preferably, the recombinant chimeric antigen comprises the amino acid sequence shown in SEQ ID NO: 4;

[0039] In a specific embodiment, the two A35R proteins or antigenic fragments thereof in the recombinant chimeric antigen can dimerize under appropriate conditions to form a stable single-chain dimer structure. Therefore, in a specific embodiment, the recombinant chimeric antigen can be a single-chain dimer structure.

[0040] In addition, 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: 5;

[0041] Preferably, the C-terminus of the recombinant chimeric antigen further comprises a tag sequence; optionally, the tag is selected from at least one of a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and a SUMO tag; preferably, the tag is a His tag.

[0042] In the present invention, in order to take into account the immune protection effects against both EEV and IMV virus particles, a recombinant chimeric antigen was designed that contains both the neutralizing antigen A35 of EEV and the neutralizing antigen M1 of IMV. The two neutralizing antigens are encoded by the A35R gene and M1R gene of monkeypox virus, respectively, and are homologous genes to the A33R and L1R genes of vaccinia virus.

[0043] In a second aspect, the present invention provides a method for preparing the recombinant chimeric antigen as described in the first aspect, comprising the following steps:

[0044] A Kozak sequence and a signal peptide coding sequence are added to the 5' end of the nucleotide sequence encoding the recombinant chimeric antigen as described in the first aspect above, and a histidine tag coding sequence and a stop codon are added to the 3' end. Cloning and expression are performed, and the correct recombinants are screened. They are then transfected into expression system cells for expression, and the cell culture supernatant is collected to isolate the recombinant chimeric antigen.

[0045] In a feasible implementation of the above preparation method, the cells of the expression system are mammalian cells, insect cells, yeast cells or bacterial cells;

[0046] 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;

[0047] Optionally, the insect cells are sf9 cells, Hi5 cells, sf21 cells or S2 cells;

[0048] Optionally, the yeast cell is a Pichia pastoris cell or a yeast cell modified therefrom;

[0049] Optionally, the bacterial cells are Escherichia coli cells.

[0050] In a third aspect, the present invention provides use of the recombinant chimeric antigen as described in the first aspect above in the preparation of a medicament for preventing and / or treating poxvirus infection.

[0051] Optionally, the poxvirus is selected from the group consisting of monkeypox virus, cowpox virus, smallpox virus and vaccinia virus;

[0052] Optionally, the drug is a vaccine, preferably a recombinant protein vaccine; further preferably, the recombinant protein vaccine uses an adjuvant selected from the following: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant;

[0053] Optionally, the vaccine is in the form of a nasal spray, oral formulation, suppository or parenteral formulation;

[0054] Preferably, the nasal spray is selected from aerosols, sprays and powder sprays;

[0055] Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated preparations and pastes; further preferably, the tablets are sublingual tablets; further preferably, the granules are fine granules; further preferably, the powders are powders; further preferably, the pills are pellets;

[0056] Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, a liquid for external use, or an injectable preparation; further preferably, the injectable preparation is a push-in preparation.

[0057] In a fourth aspect, the present invention provides a vaccine or immunogenic composition comprising the recombinant chimeric antigen as described in the first aspect above, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

[0058] In some preferred embodiments, the vaccine or immunogenic composition is a poxvirus recombinant protein vaccine, which comprises the recombinant chimeric antigen and an adjuvant as described in the first aspect above;

[0059] Preferably, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant.

[0060] In a feasible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, an oral formulation, a suppository or a parenteral formulation;

[0061] Preferably, the nasal spray is selected from aerosols, sprays and powder sprays;

[0062] Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film coatings and ointments;

[0063] Further preferably, the tablet is a sublingual tablet;

[0064] Further preferably, the granules are fine granules;

[0065] Further preferably, the powder is a powder;

[0066] Further preferably, the pills are pellets;

[0067] Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, a liquid for external use, or an injectable preparation; further preferably, the injectable preparation is a push-in preparation.

[0068] In a fifth aspect, the present invention provides a method for preventing and / or treating poxvirus infection, the method comprising: administering to a subject in need thereof a preventive and / or therapeutically effective amount of the following substances: the recombinant chimeric antigen as described in the first aspect above, and / or the vaccine or immunogenic composition as described in the fourth aspect above.

[0069] The "preventively and / or therapeutically effective amount" may vary depending on the subject of administration, the subject organ, symptoms, the method of administration, etc., and can be determined based on the doctor's judgment, taking into account the type of dosage form, the method of administration, the patient's age and weight, the patient's symptoms, etc.

[0070] Beneficial effects

[0071] The inventors of the present invention have designed a recombinant chimeric antigen against poxvirus (particularly monkeypox virus), which comprises two immunogens arranged in a specific manner: monkeypox virus A35 protein or its antigenic fragment (or its derivative peptide segment), and monkeypox virus M1 protein or its antigenic fragment (or its derivative peptide segment); wherein the former is a neutralizing antigen specific to intracellular mature virus particles (IMV), and the latter is a neutralizing antigen specific to extracellular enveloped virus particles (EEV); a vaccine comprising both can stimulate an immune response against both infectious virus particles.

[0072] In a specific embodiment, the present invention forms a single-chain tandem fusion multivalent antigen by directly tandemly linking two monkeypox virus A35 proteins or antigenic fragments thereof with two monkeypox virus M1 proteins or antigenic fragments thereof, or by tandemly linking them via an appropriate linker sequence. In this tandem fusion multivalent antigen, A35 forms a stable dimer, reversing the problem of the lack of immunogenicity of the A35 protein when immunized alone. At the same time, the divalency of the M1 antigen greatly improves its specific antibody stimulation level. Compared with immunization with the two proteins alone, the tandem fusion multivalent antigen not only retains the antigenicity of each antigen, but also more efficiently activates specific protective antibodies against monkeypox virus.

[0073] Compared with poxvirus vaccines in the prior art, the vaccine products based on the recombinant chimeric antigen of the present invention have the following advantages:

[0074] 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 the advantages of low production cost, rapid response and production capacity support. Experimental verification shows that the poxvirus vaccine of the present invention has good effectiveness.

[0075] 2) Utilizing the antigenic sequences of the monkeypox virus itself, the vaccine has a high specificity for the monkeypox virus. Existing live virus vaccines are all developed based on vaccinia virus. Although vaccinia virus and monkeypox virus belong to the same Poxviridae family, there are still certain differences in their neutralizing antigenic sequences and antigenic epitopes. Therefore, their protective effects against monkeypox virus have yet to be clarified. The poxvirus vaccine of the present invention is developed based on the antigenic epitopes of monkeypox virus and therefore has a high specificity for the prevention and treatment of monkeypox virus.

[0076] 3) Monkeypox virus contains a wide variety of proteins, the vast majority of which are ineffective components that cannot stimulate an effective antiviral immune response. In addition, some viral proteins have immunosuppressive effects. Existing live virus vaccines are unable to remove these ineffective and harmful components, thus posing vaccination risks and uncertainties. The poxvirus vaccine of the present invention retains only two virus-neutralizing antigens. Experimental data show that these two antigens alone can demonstrate complete protection in mouse models. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0078] Figure 1 Shown are the gel filtration chromatography elution curves and SDS-PAGE identification results of the immunogenic proteins AA and DAM expressed in Example 2 of the present invention.

[0079] Figure 2 Shown are 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.

[0080] Figure 3 The results of affinity analysis of the immunogenic proteins M1 (A) and DAM (B) tested in Example 3 of the present invention binding to the neutralizing antibody 7D11 are shown.

[0081] Figure 4 The results of affinity analysis of the immunogenic proteins A35 (A), AA (B) and DAM (C) tested in Example 3 of the present invention binding to the neutralizing antibody A27D7 are shown.

[0082] Figure 5 Schematic diagram of the mouse immunization and virus attack strategy used in Examples 4 and 7 of the present invention.

[0083] Figure 6 The specific binding antibody titers in the serum of the immune mice detected in Example 5 of the present invention are shown, wherein, Figure 6 A is the specific antibody level against each immunogen, Figure 6 B is the level of specific antibodies against M1 and A35 epitopes.

[0084] Figure 7 The neutralizing antibody titer of the immune mouse serum tested in Example 6 of the present invention against the live VACV virus is shown.

[0085] Figure 8The figures show the preliminary experimental results of BALB / c mice infected with different doses of VACV-WR virus by intranasal drops as described in Example 7 of the present invention, wherein the horizontal axis shows the number of days after infection and the vertical axis shows the survival percentage of the mice.

[0086] Figure 9 The protective effect of each immunogenic protein described in Example 7 of the present invention on BALB / c mice challenged with VACV-WR virus intranasally is shown, wherein the horizontal axis shows the number of days after the challenge, and the vertical axis shows the percentage of weight change (A) and survival rate (B) of the mice. DETAILED DESCRIPTION

[0087] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0088] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present invention can be practiced without certain specific details. In some embodiments, raw materials, components, methods, means, etc. that are well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0089] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0090] Example 1: Immunogen Design

[0091] In this embodiment, as a representative example of the present invention, two EEV neutralizing antigens A35 and two IMV neutralizing antigens M1 are arranged in series in the order of A35-M1-A35-M1 from N-terminus to C-terminus. The resulting single-chain fusion peptide can form a single-chain four-subunit structure of A35-A35 dimerization, hereinafter referred to as DAM, which represents the recombinant chimeric antigen of the present invention.

[0092] The following sequences all use the sequence of the monkeypox virus isolate MPXV_USA_2022_MA001, and the GenBank number of the complete genome of the virus is ON563414.3.

[0093] In the DAM, the two A35 neutralizing antigens use different A35 protein fragments, one is the S90-T181 peptide segment of the A35 protein (its amino acid sequence is shown in SEQ ID NO: 1), and the other is the S64-T181 peptide segment of the A35R protein (its amino acid sequence is shown in SEQ ID NO: 2). The amino acid sequences of the two M1 neutralizing antigens are the same, both of which are shown in SEQ ID NO: 3; therefore, the amino acid sequence of the DAM is shown in SEQ ID NO: 4.

[0094] During the construction of the DAM, for protein expression and purification purposes, the inventors also added a signal peptide sequence (as shown in SEQ ID NO: 5) to the N-terminus of the DAM amino acid sequence shown in SEQ ID NO: 4, and a six-histidine tag to the C-terminus, thereby forming the complete construct shown in SEQ ID NO: 6:

[0095] DAM complete construct (SEQ ID NO: 6)

[0096]

[0097] Among them, the first underlined part is the signal peptide sequence, the bold part is the amino acid sequence of the two A35 peptide segments, the italic part is the amino acid sequence of the two M1 peptide segments, and the last underlined part is the histidine tag sequence.

[0098] In addition, for comparison, this example also designed an A35-A35 single-chain dimer structure consisting only of two EEV neutralizing antigens A35 connected in series, hereinafter referred to as AA. In the AA construction, except for not containing the two M1 peptide segments, the remaining sequence is the same as the above-mentioned DAM construction. Similarly, for the needs of protein expression and purification, the inventors also added a signal peptide sequence (as shown in SEQ ID NO: 5) at its N-terminus and a 6-histidine tag at its C-terminus, thereby forming the complete construct shown in SEQ ID NO: 7 below:

[0099] AA complete construct (SEQ ID NO: 7)

[0100]

[0101] Among them, the first underlined part is the signal peptide sequence, the bold part is the amino acid sequence of the first A35 peptide segment, the italic part is the amino acid sequence of the second A35 peptide segment, and the last underlined part is the histidine tag sequence.

[0102] In addition, for comparison, this example also designed expression constructs of single antigen M1 and A35 peptides. The amino acid sequences of M1 and A35 peptides are as follows:

[0103] Amino acid sequence of the M1 peptide (SEQ ID NO: 8):

[0104] MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKN

[0105] MCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYV

[0106] KQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAG;

[0107] Amino acid sequence of A35 peptide (SEQ ID NO: 9):

[0108] MSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT.

[0109] Example 2: Expression and purification of immunogenic proteins

[0110] Expression and purification of constructs DAM and AA

[0111] The amino acid sequences of the constructs DAM and AA designed in Example 1 were optimized for human codons to obtain nucleotide sequences encoding DAM and AA antigens, as shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively. A Kozak sequence (GCCACC) was added to the 5' end of these nucleotide sequences, and a translation stop 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 expression plasmids for the constructs DAM and AA.

[0112] The above-mentioned DAM and AA expression plasmids were transfected into 293F cells for in vitro recombinant expression. 5-7 days after transfection, the cell supernatant containing the expressed immunogen protein was collected. Then, the protein was purified. Specifically, the cell supernatant containing the immunogen protein was subjected to nickel ion affinity chromatography (HisTrap TM HP (GE)) and then further purified by gel filtration chromatography column Superdex200 10 / 300GL (GE)). Finally, the protein purity and molecular weight were identified by SDS-PAGE.

[0113] The elution curve of gel filtration chromatography and SDS-PAGE identification results are as follows Figure 1 As shown, Figure 1 It was shown that after two-step purification, highly pure AA and DAM proteins were obtained with molecular weights of ∼25 kDa and ∼75 kDa, respectively, which was in line with expectations.

[0114] Expression and purification of single immunogens M1 and A35

[0115] The amino acid sequences of the single antigen M1 and A35 peptides designed in Example 1 were human codon optimized to obtain nucleotide sequences encoding the M1 and A35 peptides, as shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively. A translation stop codon was added to the 3' end of these nucleotide sequences, and then these DNA fragments were artificially synthesized and cloned into the pET-28a expression vector to obtain expression plasmids for M1 and A35.

[0116] The expression plasmids of M1 and A35 were expressed in Escherichia coli (E. coli) system, and the formed inclusion bodies were renatured in vitro to active proteins M1 and A35 by arginine dilution renaturation method; then, the renatured M1 or A35 protein was purified by gel filtration chromatography column Superdex200 100 / 300GL, and finally, the protein purity and molecular weight were identified by SDS-PAGE.

[0117] The elution curve of gel filtration chromatography and SDS-PAGE identification results are as follows Figure 2 As shown by Figure 2 It can be seen that after the above-mentioned gel filtration chromatography purification, highly pure M1 and A35 proteins can be obtained; among them, the peak position of the M1 protein and SDS-PAGE both showed ~20 kDa, which was in line with expectations; the peak position of the A35 protein corresponded to a molecular weight of 25 kDa, while SDS-PAGE showed ~12 kDa. It can be seen that the A35 protein is a dimer formed by intermolecular interactions, which is consistent with previous literature reports.

[0118] Example 3: Antigenicity Detection of Immunogens

[0119] In order to detect the exposure and antigenicity of each antigenic epitope of the recombinant chimeric antigen of the present invention, in this example, the ability of the immunogenic proteins expressed and purified in Example 2 (including single-chain fusion immunogenic proteins DAM and AA and single antigenic proteins M1 and A35) to bind to neutralizing antibodies 7D11 and A27D7 was tested by surface plasmon resonance (SPR) experimental methods; 7D11 and A27D7 antibodies are neutralizing antibodies against vaccinia virus antigens L1 and A33, respectively. Data show that they can cross-recognize the M1 and A35 antigens of monkeypox virus, among which antibody A27D7 can recognize the dimer epitope of A35.

[0120] The results are as follows Figure 3 and Figure 4 shown.

[0121] Depend on Figure 3 As shown in Figures A and 3B, the affinity of the single antigen M1 for the neutralizing antibody 7D11 is 3.3 nM, exhibiting a slow-on and slow-off binding pattern. The affinity of the recombinant chimeric antigen DAM of the present invention for 7D11 is three times higher than that of M1, as evidenced by an increased association rate (kon) and a decreased dissociation rate (koff). This indicates that the bivalent M1 epitopes on DAM are exposed and can be recognized by specific antibodies. Furthermore, the bivalency of M1 increases the number of M1 epitopes on DAM, thereby enhancing the antibody's binding ability and antigenicity.

[0122] Depend on Figure 4 AC shows that the affinity of single antigen A35 to antibody A27D7 is 2.2 μM, showing a fast association and fast dissociation binding mode; the affinity of single-chain fusion immunogen AA to antibody A27D7 is similar to that of single antigen A35; the affinity of the recombinant chimeric antigen DAM of the present invention to antibody A27D7 is similar to the above two, indicating that the A35 epitope on DAM is exposed and presents a stable dimer form.

[0123] The above data indicate that the A35 and M1 epitopes on the recombinant chimeric antigen DAM of the present invention are well exposed and have high antigenicity; in particular, the bivalency of the M1 epitope greatly improves its antigenicity compared with the single antigen protein M1.

[0124] Example 4: Mouse immunization experiment

[0125] In order to verify the immune protection efficacy of the recombinant chimeric antigen of the present invention, the inventors respectively mixed the immunogenic proteins obtained in Example 2 with AddaVax TM Adjuvants are mixed and emulsified according to Figure 5 The replication-competent vaccinia virus Tiantan strain (VACV-VTT) was used as the attenuated live vaccine control group.

[0126] In the immunization experiments, all mice used were female BALB / c mice, aged 6-8 weeks, with an average weight of 15-20 g. Each experimental group used 6 mice. Figure 4 As shown, mice were immunized on day 0, day 21, and day 42, for a total of three times, with each dose of 10 μg per mouse; the vaccination method was intramuscular injection, the injection site was located in the thigh of the mouse, and 50 μL was injected into each leg.

[0127] VACV-VTT was used to immunize mice by scarification at the base of the tail. Specifically, the immunization was performed on day 0, with a total immunization dose of 10 7 PFU / mouse.

[0128] On the second and third immunizations, and two days before the challenge (i.e., days 19, 40, and 54), orbital blood was collected from all groups of mice. After static clotting, the blood was centrifuged at 1500 rpm for 10 minutes to obtain mouse serum. The mouse serum was immediately aliquoted and stored in a -80°C refrigerator for subsequent ELISA testing of specific antibody titers and live virus neutralization ability assays.

[0129] Example 5: ELISA test to detect the specific antibody titer induced by the vaccine

[0130] Each immunogen protein used for mouse immunization (i.e., single antigen proteins A35 and M1, single-chain fusion immunogens AA and DAM, prepared in Example 2) was diluted to 2 μg / mL with ELISA coating solution (Solebol, C1050). 100 μL of the diluted immunogen protein or cell lysate infected with the vaccinia Tiantan strain (for antibody titer detection in the attenuated live vaccine immunization group) was added to each well of a 96-well ELISA plate (Corning, 3590) and incubated at 4°C overnight. The coating solution was discarded, PBS was added to wash away the residual coating solution, and 100 μL of ELISA blocking solution (10% skim milk powder prepared in PBST) was added. The plate was incubated at room temperature for 1 hour for blocking. During the blocking period, the immune mouse serum was diluted with ELISA blocking solution, starting from 200 times and then diluted 3 times, with 11 dilutions for each sample; after the blocking was completed, the blocking solution was removed and the immune mouse serum diluted 10 times with blocking solution was added to the ELISA plate, 100 μL for each dilution, incubated at room temperature for 1 hour, and then washed 3 times with PBST; then, HRP-labeled goat anti-mouse secondary antibody (Abcam, ab6789) diluted 1:4000 with blocking solution was added, incubated at room temperature for 1 hour, and then washed 5-6 times with PBST, TMB color development solution was added for color development, and 2M hydrochloric acid was added after the reaction for an appropriate time to terminate the reaction. OD was detected on the microplate reader. 450Read the sample. An OD450 value greater than 2.5 times that of the negative control is considered positive, and the highest dilution of the serum that is considered positive is defined as the serum antibody titer (Endpoint titer). When the reaction value at the lowest dilution is still less than 2.5 times that of the negative control, the titer of the sample is defined as half of the lowest dilution, i.e., Log10 = 1.

[0131] The results of serum specific antibody titer test of mice immunized with each immunogen protein are as follows Figure 6 As shown, Figure 6 A is the specific antibody level against each immunogen, Figure 6 B is the level of specific antibodies against M1 and A35 epitopes; Figure 6 A It can be seen that the single-chain fusion antigen DAM of the present invention can well stimulate specific antibodies after the first immunization, and the specific antibody titer after each immunization is better than all control groups including the attenuated live vaccine immunization; It is worth mentioning that with respect to the A35 epitope, the A35 protein alone cannot stimulate specific antibodies and lacks immunogenicity, while the single-chain fusion modified AA and DAM can effectively stimulate specific antibody response against the A35 epitope and has good immunogenicity ( Figure 6 B); Moreover, DAM can simultaneously stimulate high levels of specific antibody responses against A35 and M1 epitopes, and both are higher than those of the single immunization group (AA and M1, Figure 6 B);

[0132] Example 6: Determination of VACV live virus neutralization ability of immune serum

[0133] Vaccinia virus (VACV) is a model virus of the poxvirus genus. Due to the high homology among the primary immunogens of the poxvirus genus, VACV, with its lower biosafety rating and ease of handling, is commonly used internationally for evaluating the neutralization ability of other poxvirus vaccines at the cellular level and for evaluating protective efficacy in mouse models. Therefore, in this example, the commonly used mouse-adapted vaccinia virus strain Western Reserve (VACV-WR) was used to determine the live virus neutralization ability of each immunogen protein at the cellular level.

[0134] The immune mouse serum obtained in Example 4 was diluted with culture medium (DMEM) containing 2% inactivated serum, and 2-fold serial dilutions were performed starting from 20-fold, with 10 dilutions per sample. VACV-WR virus was also diluted to 500 PFU / mL using the same diluent. 200 μL of diluted immune mouse serum (the diluent without immune mouse serum was used as a control well) was mixed with 200 μL of diluted virus solution and incubated at 37°C for 1 hour. Vero cells were seeded in 12-well plates one day in advance, and the optimal density was approximately 95% on the second day. The culture medium in the 12-well plates was discarded, and after washing away any remaining culture medium with PBS, the incubated serum-virus mixture was added to the 12-well plates and infected at 37°C for 2 hours. After infection, the virus-serum mixture was removed, any remaining virus was washed away with PBS, and a prepared carboxymethylcellulose-DMEM mixture (2% carboxymethylcellulose and 2×DMEM, mixed in a 1:1 ratio) was added and incubated at 37°C for 48-60 hours. After obvious CPE was observed under a microscope, 4% paraformaldehyde fixative was added and fixed at room temperature for 2 hours, followed by crystal violet staining and counting.

[0135] PRNT 50 The calculation method is as follows: the well containing only virus is used as the control well, and the number of plaques counted in all experimental wells is divided by the number of plaques in the control well, which is the inhibition rate of the serum against the virus in each experimental well. Then, PRNT is calculated using the log (inhibitor) vs. normalized response--Variable slope formula in GraphPad software. 50 .

[0136] See the results Figure 7 .

[0137] Because the live virus neutralization experiment used the VACV-WR strain, which hardly produces EEV, we used IMV in our neutralization experiments.

[0138] like Figure 7 As shown in the figure, since A35 does not exist on IMV virus particles, the A35 and AA immunization groups cannot show neutralization ability at the neutralization level of live virus; while the M1, DAM and VACV-VTT immunization groups can all produce neutralizing antibodies against IMV virus particles, among which the neutralizing antibody stimulation ability of DAM is significantly higher than that of the M1 and attenuated live vaccine VACV-VTT immunization groups.

[0139] Example 7: Challenge protection experiment

[0140] When the VACV-WR virus strain is intranasally infected with BALB / c mice, it can cause the mice to die. To determine the appropriate challenge dose, we first conducted a preliminary experiment of intranasal infection with different doses of VACV-WR virus on BALB / c mice aged 17-19 weeks (the same age as when challenged after three immunizations) to determine the LD50 of VACV-WR infection in BALB / c mice under our experimental system. The preliminary experimental results are shown in Figure 2. Figure 8 As shown, when the challenge dose was 2×10 5 When the virus was detected at 100 PFU, all mice died within 7 days after infection. By calculation, the amount of virus at this time was 7LD50, which we subsequently used as the challenge dose for animal experiments.

[0141] like Figure 5 As shown in Example 4, mice immunized three times were challenged with 7LD50 VACV-WR virus intranasally two weeks after the third immunization, i.e., on the 56th day, to evaluate the protective effect of the vaccine in the animal model. The weight change percentage and survival percentage of mice in each challenge experimental group are shown in Figure 4. Figure 9 A and Figure 9 B.

[0142] Figure 9 The results of A and 9B showed that the single antigen A35 immunization group had no protective effect on mice infected with the VACV-WR virus, and all mice died 6 days after the infection, which was consistent with the result that they could not effectively stimulate specific antibodies; although the single antigen M1 and the single-chain fusion immunogen AA showed a higher level of specific antibody levels in Example 4 and a higher level of neutralizing antibody stimulation ability in Example 5, they could only protect 60% of the mice from VACV-WR infection and death, and the surviving mice also showed a large degree of weight loss; compared with the above groups, the single-chain fusion immunogen DAM of the present invention can have a 100% protective effect on VACV-WR infected mice, and no obvious weight changes occurred.

[0143] The above results show that the single-chain fusion immunogen DAM of the present invention exhibits excellent protective effect in the mouse model.

[0144] 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the present invention.

[0145] Sequences involved in this application:

[0146] SEQ ID NO: 1 (amino acid sequence of the first A35 peptide segment in DAM)

[0147]

[0148] SEQ ID NO: 2 (amino acid sequence of the second A35 peptide segment in DAM)

[0149]

[0150] SEQ ID NO: 3 (amino acid sequence of the M1 peptide segment in DAM)

[0151] SEQ ID NO: 4 (amino acid sequence of DAM)

[0152]

[0153] SEQ ID NO: 5 (amino acid sequence of signal peptide)

[0154]

[0155] SEQ ID NO: 6 (amino acid sequence of the complete DAM construct in Example 1)

[0156]

[0157] SEQ ID NO: 7 (amino acid sequence of the complete AA construct in Example 1)

[0158]

[0159] SEQ ID NO: 8 (amino acid sequence of the single antigen M1 peptide segment in Example 1)

[0160] MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCS

[0161] ADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQT

[0162] CNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQI

[0163] APRQVAG

[0164] SEQ ID NO: 9 (amino acid sequence of the single antigen A35 peptide segment in Example 1)

[0165] MSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLI

[0166] DYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT

[0167] SEQ ID NO: 10 (nucleotide sequence encoding the complete DAM construct shown in SEQ ID NO: 6)

[0168]

[0169]

[0170]

[0171] SEQ ID NO: 11 (nucleotide sequence encoding the complete AA construct shown in SEQ ID NO: 7)

[0172]

[0173] SEQ ID NO: 12 (nucleotide sequence encoding a single antigen M1 peptide segment (i.e., SEQ ID NO: 8))

[0174] atgggagcagctgcgtcaatacaaacaactgtaaacaccctgagcgaacgtattagctccaaacttgagcaagaggcaaacgcgagc

[0175] gcgcaaacgaaatgcgatattgagatcggcaacttctatatccgccaaaatcacggttgtaatattaccgtcaagaacatgtgcagcgcg

[0176] gacgcggacgcgcagctggacgccgttttgtctgcagcgaccgaaacctattccggtctgaccccggagcagaaagcgtacgttccg

[0177] gctatgttcaccgcagcactcaatatccaaaccagcgtcaataccgttgttcgtgattttgaaaattacgtgaagcagacgtgcaactcct

[0178] cggcggtggtggataacaaactgaagatccaaaacgtgattatcgacgaatgttacggcgctccgggttctccgaccaacttggagttt

[0179] atcaacactggcagcagcaaaggcaactgcgctattaaggcgctgatgcagctgactacaaaggcgaccacgcagatcgccccacg

[0180] tcaggtggccggt

[0181] SEQ ID NO:13 (Nucleotide sequence encoding the single antigen A35 peptide segment (i.e., SEQ ID NO:9))

[0182] atgtcaactacacaatatgatcacaaagaaagttgcaacggcttatattatcagggttcttgttatatcctgcacagcgactacaagtctttt

[0183] gaggatgcgaaagctaactgcgcggcagaaagcagcaccctgccgaataagtccgacgtgttgaccacgtggctgatcgattacgtg

[0184] gaagatacctggggttccgacggcaacccgattaccaaaactacgagcgactaccaggatagcgacgtttcgcaagaggttcgtaaat

[0185] acttctgcacc

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 according to the following formula (I): A1-C1-M1-C2-A2-C3-M2 (I)； In formula (I): A1 represents the antigenic fragment I of the monkeypox virus A35 protein, the amino acid sequence of which is shown in SEQ ID NO: 1; A2 represents antigenic fragment II of the monkeypox virus A35 protein, whose amino acid sequence is: i) the amino acid sequence set forth in SEQ ID NO: 1, or ii) the amino acid sequence set forth in SEQ ID NO: 1 plus an amino acid sequence formed by extending the amino acid sequence thereof by 1 to 30 amino acids toward the N-terminus of the A35 protein; M1 and M2 are the same, both representing antigenic fragments of the monkeypox virus M1 protein, and their amino acid sequences are shown in SEQ ID NO: 3; C1, C2, and C3 are each independently none or a linker sequence (GGGGS)n, wherein n is any integer between 1 and 10.

2. The poxvirus recombinant chimeric antigen according to claim 1, characterized in that Regarding item A2 ii), the amino acid sequence of SEQ ID NO: 1 plus a fragment extending 1-30 amino acids to the N-terminus of the A35 protein is shown in SEQ ID NO:

2.

3. The poxvirus recombinant chimeric antigen according to claim 1, characterized in that The A1 represents the amino acid sequence shown in SEQ ID NO: 1, the A2 represents the amino acid sequence shown in SEQ ID NO: 2, and the M1 and M2 are the same, both representing the amino acid sequence shown in SEQ ID NO:

3.

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

4.

5. A poxvirus recombinant chimeric antigen, characterized in that: The poxvirus recombinant chimeric antigen consists of the following sequences from N-terminus to C-terminus: a signal peptide sequence, the amino acid sequence of the poxvirus recombinant chimeric antigen according to any one of claims 1 to 4, and a tag sequence.

6. The poxvirus recombinant chimeric antigen according to claim 5, characterized in that The signal peptide sequence is shown in SEQ ID NO: 5; And / or, the tag is at least one selected from Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and SUMO tag.

7. The poxvirus recombinant chimeric antigen according to claim 6, characterized in that The tag is a His tag.

8. The poxvirus recombinant chimeric antigen according to claim 5, characterized in that The amino acid sequence of the poxvirus recombinant chimeric antigen is shown in SEQ ID NO:

6.

9. The poxvirus recombinant chimeric antigen according to any one of claims 1 to 8, characterized in that The poxvirus recombinant chimeric antigen is a single-chain dimer structure.

10. A method for preparing the poxvirus recombinant chimeric antigen according to any one of claims 1 to 4, comprising the following steps: A Kozak sequence and a signal peptide coding sequence are added to the 5' end of the nucleotide sequence encoding the poxvirus recombinant chimeric antigen according to any one of claims 1 to 4, and a histidine tag coding sequence and a stop codon are added to the 3' end. Cloning and expression are performed, and the correct recombinant is screened. It is then transfected into expression system cells for expression, and the cell culture supernatant is collected to isolate the poxvirus recombinant chimeric antigen.

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

12. The preparation method according to claim 11, characterized in that: The mammalian cells are HEK293T cells, 293F series cells or CHO cells; And / or, the insect cell is an sf9 cell, a Hi5 cell, an sf21 cell or an S2 cell; And / or, the yeast cell is a Pichia pastoris cell or a yeast cell transformed therefrom; And / or, the bacterial cells are Escherichia coli cells.

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

14. Use of the poxvirus recombinant chimeric antigen according to any one of claims 1 to 9 in the preparation of a vaccine for preventing poxvirus infection; in, The poxvirus is selected from the group consisting of monkeypox virus and / or vaccinia virus.

15. The use according to claim 14, characterized in that: The vaccine is a recombinant protein vaccine, and the recombinant protein vaccine uses an adjuvant selected from the following: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant.

16. A vaccine or immunogenic composition comprising the poxvirus recombinant chimeric antigen according to any one of claims 1 to 9, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

17. The vaccine or immunogenic composition according to claim 16, characterized in that The vaccine or immunogenic composition is a poxvirus recombinant protein vaccine, which comprises the poxvirus recombinant chimeric antigen according to any one of claims 1 to 9 and an adjuvant, wherein the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant.

18. The vaccine or immunogenic composition according to claim 16 or 17, characterized in that The vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository or parenteral formulation.

19. The vaccine or immunogenic composition according to claim 18, characterized in that The nasal spray is selected from aerosols, sprays and powder sprays; and / or, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film coatings and ointments; And / or, the parenteral preparation is a transdermal preparation, an ointment, a plaster, a liquid for external use or an injectable preparation.

20. The vaccine or immunogenic composition according to claim 19, characterized in that The tablet is a sublingual tablet; and / or, the granules are fine granules; And / or, the powder is a powder; and / or, the pills are pellets; And / or, the injectable formulation is a bolus formulation.

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