Recombinant novel coronavirus Omicron variant RBD trimer antigen, immunogenic composition containing same and application of recombinant novel coronavirus Omicron variant RBD trimer antigen

Abstract

The invention relates to a novel coronavirus recombinant chimeric antigen, a related product thereof, a preparation method and application of the novel coronavirus recombinant chimeric antigen in preparation of a novel coronavirus vaccine. The recombinant chimeric antigen is a recombinant new coronavirus Omicron variant RBD trimer antigen, various types of vaccines based on the recombinant chimeric antigen can induce strong immune response aiming at various subtype variants (including BA.1, BA.2, BA.5, XBB, EG.5, BA.2. 86, JN.1, KP.2 and the like) of the new coronavirus Omicron, and particularly, compared with a protein vaccine of the recombinant chimeric antigen, the recombinant chimeric antigen can induce strong immune response. The mRNA vaccine based on the recombinant chimeric antigen can induce an organism to generate obviously higher RBD specific binding antibody level and neutralizing antibody level, so that the mRNA vaccine can be used as a candidate of a new generation of new coronavirus vaccines, and has potential clinical application value and prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to a new coronavirus recombinant chimeric antigen, its related products, preparation method and application in the preparation of new coronavirus vaccine. Background Art

[0002] The new coronavirus (SARS-CoV-2) belongs to the genus Betacoronavirus in the family Coronaviridae. It is a single-stranded positive-sense RNA enveloped virus and a zoonotic virus that can widely infect humans and a variety of animals. The most common symptoms of SARS-CoV-2 coronavirus infection are fever, chills, and sore throat, but there are a range of other symptoms. SARS-CoV-2 vaccines are effective in preventing severe illness and death. Although people can still be infected with SARS-CoV-2 after being vaccinated, they are more likely to have mild symptoms or no symptoms.

[0003] The spike protein (S) on the surface of the SARS-CoV-2 envelope mediates the virus' attachment, fusion, and entry into host cells. The receptor binding domain (RBD) located at the C-terminus of the S protein is considered to be the most important target region for inducing the body to produce neutralizing antibodies, and is also an important target for vaccine development. As a vaccine immunogen, RBD can stimulate the body to produce neutralizing antibodies, which can effectively inhibit the binding of the virus to the receptor, thereby inhibiting the virus from infecting and invading host cells. Therefore, RBD is also regarded as an important target for the development of new crown vaccines.

[0004] SARS-CoV-2 has evolved into a variety of epidemic variants, mainly including alpha, beta, gamma, delta and other variants. The Omicron strain has also evolved into a series of variants, including BA.1, BA.2, BA.2.75, BA.4 / 5, BQ.1.1, BF.7, XBB, XBB.1.5, XBB.1.16, EG.5, JN.1, BA.2.86, KP.2, etc. These newly emerged Omicron epidemic variants have a large number of mutation sites in their S protein, and have serious immune escape from the humoral immune response activated by existing new coronavirus neutralizing antibody drugs and vaccines. Therefore, the need to develop a new generation of new coronavirus vaccines that can cope with a variety of epidemic variants is urgent, and the neutralization effect against Omicron and other variants is an important indicator of the new generation of new coronavirus vaccines. Summary of the invention

[0005] In order to overcome the problems existing in the above-mentioned prior art, the present invention provides a recombinant chimeric antigen of the new coronavirus, its related products, preparation method and application in the preparation of new coronavirus vaccine.

[0006] The recombinant chimeric antigen is the RBD trimer antigen of the recombinant novel coronavirus Omicron (also referred to as "Omicron" in this article). The recombinant protein vaccine or various nucleic acid vaccines (especially the latter) based on the recombinant chimeric antigen or its encoding nucleic acid can stimulate a strong immune response in a balanced manner against the various subtype variants of the novel coronavirus Omicron, and has a broad-spectrum and strong immunogenicity.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In the first aspect, the present invention provides a recombinant chimeric antigen of a novel coronavirus, wherein the recombinant chimeric antigen has an amino acid sequence as shown in formula (I):

[0009] (AB)-C1-(A-B')-C2-(AB”)

[0010] (I)

[0011] In formula (I):

[0012] AB represents the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Omicron variant XBB.1.5 subtype or a portion 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;

[0013] A-B' represents the amino acid sequence of the RBD domain of the S protein of the EG.5 subtype of the novel coronavirus Omicron variant or a portion thereof, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and has the same or substantially the same immunogenicity thereto;

[0014] "AB" means the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Omicron variant BA.2.86 subtype or a portion 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;

[0015] C1 and C2 are the same or different, and each independently represents a linker (GGS) n ; Wherein, n=0,1,2,3,4 or 5.

[0016] For the above recombinant chimeric antigens:

[0017] Preferably, a portion of the RBD domain of the S protein of the novel coronavirus Omicron variant XBB.1.5 subtype is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of its entire amino acid sequence;

[0018] Preferably, a portion of the RBD domain of the S protein of the novel coronavirus Omicron variant EG.5 subtype is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of its entire amino acid sequence;

[0019] Preferably, a portion of the RBD domain of the S protein of the novel coronavirus Omicron variant BA.2.86 subtype is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of its entire amino acid sequence;

[0020] Preferably, n=0, 1, 2 or 3.

[0021] In some preferred embodiments, the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Omicron variant XBB.1.5 subtype or a portion thereof is as shown in SEQ ID NO: 1, or an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1, and having the same or substantially the same immunogenicity as the amino acid sequence;

[0022] And / or, the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Omicron variant EG.5 subtype or a portion thereof is as shown in SEQ ID NO: 2, or an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2, and having the same or substantially the same immunogenicity as that thereof;

[0023] And / or, the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Omicron variant BA.2.86 subtype or a portion thereof is as shown in SEQ ID NO: 3, or an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 3, and having the same or substantially the same immunogenicity as that thereof;

[0024] And / or, n=0, 1 or 2.

[0025] In a further preferred embodiment, AB represents the amino acid sequence shown in SEQ ID NO: 1, A-B' represents the amino acid sequence shown in SEQ ID NO: 2, and AB" represents the amino acid sequence shown in SEQ ID NO: 3;

[0026] Further preferably, the recombinant chimeric antigen comprises the amino acid sequence shown in SEQ ID NO:4.

[0027] In a feasible embodiment, the N-terminus of the recombinant chimeric antigen further includes a signal peptide sequence, preferably, the signal peptide sequence is as shown in SEQ ID NO: 5; and / or, the C-terminus of the recombinant chimeric antigen further includes a tag sequence, preferably, the tag is a 6×His tag. In a feasible specific embodiment, the recombinant chimeric antigen includes an amino acid sequence as shown in SEQ ID NO: 6.

[0028] 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:

[0029] A Kozak sequence and optionally a coding sequence of a signal peptide are added to the 5' end of the nucleotide sequence encoding the recombinant chimeric antigen as described above, and an optional coding sequence of a histidine tag and a stop codon are added to the 3' end, and cloned and expressed. The correct recombinant is screened, and then transfected into expression system cells for expression, and the cell culture supernatant is collected to isolate and obtain the recombinant chimeric antigen.

[0030] In a feasible embodiment, the expression system cell is a mammalian cell, an insect cell, a yeast cell or a bacterial cell;

[0031] Optionally, the mammalian cell is a HEK293T cell, a 293F series cell or a CHO cell; further optionally, the 293F series cell is a HEK293F cell, a Freestyle293F cell or an Expi293F cell;

[0032] Optionally, the insect cell is a sf9 cell, a Hi5 cell, a sf21 cell or a S2 cell;

[0033] Optionally, the yeast cell is a Pichia pastoris cell or a yeast cell transformed therefrom;

[0034] Optionally, the bacterial cells are Escherichia coli cells.

[0035] In a third aspect, the present invention provides a polynucleotide encoding the recombinant chimeric antigen as described in the first aspect above.

[0036] The polynucleotide is a nucleotide sequence optimized for human codons and may be DNA or mRNA;

[0037] In some embodiments, the polynucleotide is a DNA molecule, preferably, the DNA molecule comprises a DNA sequence as shown in SEQ ID NO:7 or 8; wherein the DNA sequence as shown in SEQ ID NO:7 encodes a recombinant chimeric antigen having an amino acid sequence as shown in SEQ ID NO:4 (excluding a signal peptide and a tag sequence), and the DNA sequence as shown in SEQ ID NO:8 encodes a recombinant chimeric antigen having an amino acid sequence as shown in SEQ ID NO:6 (including a signal peptide and a tag sequence).

[0038] In other embodiments, the polynucleotide is an mRNA molecule, preferably, the mRNA molecule includes an mRNA sequence as shown in SEQ ID NO:9 or 10; wherein the mRNA sequence as shown in SEQ ID NO:9 is the corresponding sequence of the DNA sequence as shown in SEQ ID NO:7, encoding a recombinant chimeric antigen as shown in SEQ ID NO:4 (excluding signal peptide and tag sequence); the mRNA sequence as shown in SEQ ID NO:10 is the corresponding sequence of the DNA sequence as shown in SEQ ID NO:8, encoding a recombinant chimeric antigen with an amino acid sequence as shown in SEQ ID NO:6 (including signal peptide and tag sequence).

[0039] In a 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 control element operably linked to the polynucleotide.

[0040] In a fifth aspect, the present invention provides an expression vector comprising the nucleic acid construct as described in the fourth aspect above.

[0041] In a sixth aspect, the present invention provides a host cell transformed or transfected with the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, or the expression vector as described in the fifth aspect.

[0042] In the seventh aspect, the present invention provides the use of the recombinant chimeric antigen as described in the first aspect, the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, the expression vector as described in the fifth aspect, or the host cell as described in the sixth aspect in the preparation of a vaccine for preventing and / or treating novel coronavirus infection.

[0043] Preferably, the novel coronavirus is one or more selected from the following: SARS-CoV-2 original strain, SARS-CoV-2 variant strains Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2), Omicron subtype BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4, BA.5, XBB, EG.5, BA.2.86, JN.1, KP.2;

[0044] Preferably, the vaccine is used for single immunization or sequential immunization with other types of new coronavirus vaccines; further preferably, the other types of new coronavirus vaccines are inactivated vaccines.

[0045] 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.

[0046] In a preferred embodiment, the vaccine or immunogenic composition is a novel coronavirus recombinant protein vaccine, which comprises the recombinant chimeric antigen and adjuvant as described in the first aspect above;

[0047] Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant; further optionally, the adjuvant is a MF59-like adjuvant; further optionally, the adjuvant is Addavax.

[0048] In another preferred embodiment, the vaccine or immunogenic composition is a novel coronavirus DNA vaccine comprising:

[0049] (1) a eukaryotic expression vector; and

[0050] (2) a DNA sequence encoding the recombinant chimeric antigen as described in the first aspect constructed into the eukaryotic expression vector, preferably a DNA sequence as shown in SEQ ID NO: 7 or 8;

[0051] Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

[0052] In another preferred embodiment, the vaccine or immunogenic composition is a novel coronavirus mRNA vaccine, and the mRNA vaccine comprises:

[0053] (I) an mRNA sequence encoding the recombinant chimeric antigen as described in the first aspect above, preferably an mRNA sequence as shown in SEQ ID NO: 9 or 10; and

[0054] (II) Lipid nanoparticles.

[0055] In another preferred embodiment, the vaccine or immunogenic composition is a novel coronavirus-viral vector vaccine, comprising:

[0056] (1) viral backbone vectors; and

[0057] (2) a DNA sequence encoding the recombinant chimeric antigen as described in the first aspect constructed into the viral backbone vector, preferably a DNA sequence as shown in SEQ ID NO: 7 or 8;

[0058] 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.

[0059] In another preferred embodiment, the vaccine or immunogenic composition is a novel coronavirus nanoparticle vaccine;

[0060] Preferably, the carrier of the nanoparticle is ferritin, and the recombinant chimeric antigen as described in the first aspect above is covalently linked to ferritin and self-assembled into nanoparticles, so that the recombinant chimeric antigen is presented on the surface of the nanoparticle; further preferably, the recombinant chimeric antigen is linked to ferritin via a linker, or is covalently linked via a SpyTag / SpyCatcher linking system.

[0061] In a feasible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, an oral preparation, a suppository or a parenteral preparation;

[0062] Preferably, the nasal spray is selected from aerosols, sprays and powder sprays;

[0063] Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film coatings and ointments;

[0064] Further preferably, the tablet is a sublingual tablet;

[0065] Further preferably, the granules are fine granules;

[0066] Further preferably, the powder is a powder;

[0067] Further preferably, the pills are pellets;

[0068] Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid preparation, or an injectable preparation; further preferably, the injectable preparation is a pushable preparation.

[0069] In a ninth aspect, the present invention provides a kit comprising the vaccine or immunogenic composition as described in the eighth aspect above, and optionally other types of novel coronavirus vaccines, wherein the vaccine or immunogenic composition and the other types of novel coronavirus vaccines are packaged separately;

[0070] Preferably, the other type of new coronavirus vaccine is an inactivated new coronavirus vaccine.

[0071] In the tenth aspect, the present invention provides a method for preventing and / or treating novel coronavirus infection, the method comprising: administering a preventive and / or therapeutically effective amount of the following substances to a subject in need: 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.

[0072] Optionally, the new coronavirus is one or more selected from the following: SARS-CoV-2 original strain, SARS-CoV-2 variant strains Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2), Omicron subtype BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4, BA.5, XBB, EG.5, BA.2.86, JN.1, KP.2.

[0073] 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.

[0074] Beneficial Effects

[0075] The present invention provides a recombinant chimeric antigen formed by the S protein RBD domain of the XBB.1.5 subtype, EG.5 subtype and BA.2.86 subtype of the novel coronavirus Omicron variant, a polynucleotide encoding it, and various types of vaccine products based on the recombinant chimeric antigen or its encoding nucleic acid. The vaccine product based on the recombinant chimeric antigen or its encoding nucleic acid can stimulate a strong immune response against each subtype of the novel coronavirus Omicron variant (including BA.1, BA.2, BA.5, XBB, EG.5, BA.2.86, JN.1 and KP.2, etc.), indicating that it has a good immune protection effect on each subtype of Omicron. In particular, the nucleic acid vaccine (especially mRNA vaccine) based on the encoding nucleic acid of the recombinant chimeric antigen can induce the body to produce a higher RBD-specific binding antibody titer and a neutralizing antibody titer against a variety of novel coronavirus pseudoviruses compared with its corresponding protein vaccine, and therefore, it can be used as a candidate for a new generation of novel coronavirus vaccines, with potential clinical application value and prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0077] Figure 1 The recombinant expression plasmid pCAGGS-PPP was transfected into Expi293F as described in Example 2 of the present invention. TM The molecular sieve chromatography curve of the recombinant trimeric antigen PPP expressed later and the SDS-PAGE identification results of the eluate at its elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0078] Figure 2 The recombinant expression plasmid pCAGGS-Delta-BA.2-BA.5 was transfected into Expi293F TM The molecular sieve chromatography curve of the recombinant trimeric antigen Delta-BA.2-BA.5 (abbreviated as D25) expressed later and the SDS-PAGE identification results of the eluate at its elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0079] Figure 3 The recombinant expression plasmid pCAGGS-XBB.1.5-EG.5-BA.2.86 was transfected into Expi293F TMThe molecular sieve chromatography curve of the recombinant trimeric antigen XBB.1.5-EG.5-BA.2.86 (abbreviated as XE6) expressed later and the SDS-PAGE identification results of the eluate at its elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0080] Figure 4 It is a schematic diagram of the immunization procedure and sample collection process of the mouse immunization experiment recorded in Example 3 of the present invention.

[0081] Figure 5 This is a graph of the results of detecting the RBD-specific binding antibody titer produced by mice stimulated by different recombinant trimer antigen protein vaccines after one immunization as described in Example 4 of the present invention by enzyme-linked immunosorbent assay (ELISA).

[0082] Figure 6 This is a result diagram recorded in Example 4 of the present invention, which is a result of detecting the RBD-specific binding antibody titer produced by mice stimulated by different recombinant trimeric antigen protein vaccines after secondary immunization using an enzyme-linked immunosorbent assay (ELISA).

[0083] Figure 7 It is a result diagram recorded in Example 4 of the present invention, which is a result of detecting the RBD-specific binding antibody titer produced in mice after three immunizations with different recombinant trimer antigen protein vaccines by enzyme-linked immunosorbent assay (ELISA).

[0084] Figure 8 This is a graph of the neutralizing antibody titer detection results against the prototype strain of the new coronavirus produced in the mouse serum after the third immunization of mice with different recombinant trimeric antigen protein vaccines as recorded in Example 5 of the present invention.

[0085] Fig. 9 This is a graph of the neutralizing antibody titer detection results against the Delta strain pseudovirus of the new coronavirus produced in the mouse serum after the third immunization of mice with different recombinant trimer antigen protein vaccines as recorded in Example 5 of the present invention.

[0086] Fig.10 This is a graph of the results of the neutralizing antibody titer test against the novel coronavirus Omicron BA.1 pseudovirus produced in the mouse serum after the third immunization of mice with different recombinant trimeric antigen protein vaccines as recorded in Example 5 of the present invention.

[0087] Fig.11 This is a graph of the results of the neutralizing antibody titer test against the novel coronavirus Omicron BA.2 pseudovirus produced in the mouse serum after the third immunization of mice with different recombinant trimer antigen protein vaccines as recorded in Example 5 of the present invention.

[0088] Fig.12 This is a graph of the results of the neutralizing antibody titer test against the novel coronavirus Omicron BA.5 pseudovirus produced in the mouse serum after the third immunization of mice with different recombinant trimer antigen protein vaccines as recorded in Example 5 of the present invention.

[0089] Fig.13 This is a graph of the results of the neutralizing antibody titer test against the novel coronavirus Omicron XBB pseudovirus produced in the mouse serum after the third immunization of mice with different recombinant trimer antigen protein vaccines as recorded in Example 5 of the present invention.

[0090] Fig.14 This is a graph of the results of the neutralizing antibody titer test against the novel coronavirus Omicron EG.5 pseudovirus produced in the mouse serum after the third immunization of mice with different recombinant trimer antigen protein vaccines as recorded in Example 5 of the present invention.

[0091] Fig.15 This is a graph of the results of the neutralizing antibody titer test against the novel coronavirus Omicron BA.2.86 pseudovirus produced in the mouse serum after the third immunization of mice with different recombinant trimeric antigen protein vaccines as recorded in Example 5 of the present invention.

[0092] Fig.16 This is a graph of the results of the neutralizing antibody titer test against the novel coronavirus Omicron JN.1 pseudovirus produced in the mouse serum after the third immunization of mice with different recombinant trimer antigen protein vaccines as recorded in Example 5 of the present invention.

[0093] Fig.17 This is a graph of the results of the neutralizing antibody titer test against the novel coronavirus Omicron KP.2 pseudovirus produced in the mouse serum after the third immunization of mice with different recombinant trimer antigen protein vaccines as recorded in Example 5 of the present invention.

[0094] Fig.18 It is a radar chart of the detection results of neutralizing antibody titers against different strains of pseudoviruses produced in mouse serum after the mice were immunized for the third time with different immunogens as recorded in Example 5 of the present invention.

[0095] Fig.19 This is a graph of the results of detecting the RBD-specific binding antibody titer produced by mice stimulated by the protein vaccine and mRNA vaccine of the recombinant trimer antigen XE6 after one immunization as described in Example 7 of the present invention by enzyme-linked immunosorbent assay (ELISA).

[0096] Fig. 20This is a graph of the results of detecting the RBD-specific binding antibody titer produced by the protein vaccine and mRNA vaccine of the recombinant trimer antigen XE6 in mice stimulated by the second immunization as described in Example 7 of the present invention by enzyme-linked immunosorbent assay (ELISA).

[0097] Fig.21 This is a graph of the results of detecting the RBD-specific binding antibody titer produced by mice stimulated by the protein vaccine and mRNA vaccine of the recombinant trimer antigen XE6 after three immunizations as described in Example 7 of the present invention by enzyme-linked immunosorbent assay (ELISA).

[0098] Fig. 22 This is a graph of the neutralizing antibody titer test results against the pseudovirus of the 2019-nCoV Omicron XBB.1.5 strain produced in the mouse serum by the protein vaccine and the mRNA vaccine of the recombinant trimer antigen XE6 after the second immunization of mice as recorded in Example 8 of the present invention.

[0099] Fig.23 This is a graph of the neutralizing antibody titer test results against the pseudovirus of the 2019-nCoV Omicron EG.5 strain produced in the mouse serum by the protein vaccine and the mRNA vaccine of the recombinant trimer antigen XE6 after the second immunization of mice as recorded in Example 8 of the present invention.

[0100] Fig.24 This is a graph of the neutralizing antibody titer test results against the novel coronavirus Omicron BA.2.86 strain pseudovirus produced in the mouse serum by the protein vaccine and mRNA vaccine of the recombinant trimer antigen XE6 recorded in Example 8 of the present invention after the second immunization of mice.

[0101] Fig.25 It is a radar chart of the detection results of neutralizing antibody titers against different strains of pseudoviruses produced in mouse serum after the second immunization of mice with protein vaccines and mRNA vaccines of different immunogens as recorded in Example 8 of the present invention.

[0102] Fig.26 This is a graph showing the results of the neutralizing antibody titer test against the pseudovirus of the novel coronavirus Omicron XBB.1.5 strain produced in the mouse serum by the protein vaccine and the mRNA vaccine of the recombinant trimer antigen XE6 after the third immunization of mice as recorded in Example 8 of the present invention.

[0103] Fig. 27 This is a graph of the neutralizing antibody titer test results against the novel coronavirus Omicron BA.2.86 strain pseudovirus produced in the mouse serum by the protein vaccine and mRNA vaccine of the recombinant trimer antigen XE6 recorded in Example 8 of the present invention after the third immunization of mice.

[0104] Fig.28 This is a graph showing the results of the neutralizing antibody titer test against the pseudovirus of the 2019-nCoV Omicron EG.5 strain produced in the mouse serum by the protein vaccine and the mRNA vaccine of the recombinant trimer antigen XE6 after the third immunization of mice as recorded in Example 8 of the present invention.

[0105] Fig.29 It is a radar chart of the detection results of neutralizing antibody titers against different strains of pseudoviruses produced in mouse serum after the third immunization of mice with protein vaccines and mRNA vaccines of different immunogens as recorded in Example 8 of the present invention. DETAILED DESCRIPTION

[0106] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0107] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0108] Unless explicitly 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 components.

[0109] Example 1: Design of expression constructs for SARS-CoV-2 prototype strain RBD trimer (PPP), Delta-BA.2-BA.5 trimer (D25), and XBB.1.5-EG.5-BA.2.86 RBD trimer (XE6)

[0110] In this embodiment, expression constructs of the novel coronavirus prototype strain RBD trimer (abbreviated as PPP), Delta-BA.2-BA.5 trimer (abbreviated as D25), and XBB.1.5-EG.5-BA.2.86 RBD trimer (abbreviated as XE6, representing the recombinant chimeric antigen of the present invention) were designed respectively, and the specific scheme is as follows:

[0111] (1) The sequences of the R319-K537 segment of the RBD domain of the three novel coronavirus prototype strains were directly connected in series, a signal peptide (SEQ ID NO: 5) was connected to the N-terminus, and a 6-histidine tag was added to the C-terminus to obtain the construct of the prototype strain RBD trimer PPP;

[0112] (2) The sequences of the R319-V534 segment of the RBD domain of the Delta variant of the novel coronavirus, the V320-V534 segment of the RBD domain of the BA.2 subtype of the Omicron variant, and the V320-K537 segment of the RBD domain of the BA.5 subtype of the Omicron variant were directly connected in series, a signal peptide (SEQ ID NO: 5) was connected to the N-terminus, and a 6-histidine tag was added to the C-terminus to obtain the Delta-BA.2-BA.5 trimer D25 construct;

[0113] (3) The sequence of the R319-V534 segment of the RBD domain of the XBB.1.5 subtype of the novel coronavirus Omicron variant (SEQ ID NO: 1), the sequence of the V320-V534 segment of the RBD domain of the EG.5 subtype of the novel coronavirus Omicron variant (SEQ ID NO: 2), and the sequence of the V320-K537 segment of the RBD domain of the BA.2.86 subtype of the novel coronavirus Omicron variant (SEQ ID NO: 3) were directly connected in series, a signal peptide (SEQ ID NO: 5) was connected to the N-terminus, and a 6-histidine tag was added to the C-terminus to obtain the construct of the XBB.1.5-EG.5-BA.2.86 chimeric RBD trimer antigen XE6, the amino acid sequence of which is shown in SEQ ID NO: 6.

[0114] Example 2: Expression and purification of recombinant trimeric antigen proteins PPP, D25 and XE6

[0115] Construction of recombinant expression plasmid:

[0116] The amino acid sequences of the expression constructs of the recombinant trimeric antigens PPP, D25 and XE6 designed in the above Example 1 were optimized using human codons, and the corresponding DNA coding sequences were shown as PPP (SEQ ID NO: 11), D25 (SEQ ID NO: 12), and XE6 (SEQ ID NO: 8), respectively; a stop codon was added to the 3' end of these DNA coding sequences, and a Kozak sequence gccacc was added upstream of their 5' end. These DNA sequences containing the Kozak sequence were synthesized by Nanjing GenScript Biotechnology Co., Ltd.; the synthesized DNA sequences were cloned into the pCAGGS plasmid through the EcoRI and XhoI restriction sites, respectively, to obtain recombinant expression plasmids pCAGGS-PPP, pCAGGS-Delta-BA.2-BA.5, and pCAGGS-XBB.1.5-EG.5-BA.2.86 expressing the recombinant trimeric antigens PPP, D25 and XE6, respectively.

[0117] Protein expression and purification:

[0118] Expi 293F cells were used to express single-chain heterologous RBD trimers of PPP, D25, and XE6.

[0119] The recombinant expression plasmids pCAGGS-PPP, pCAGGS-Delta-BA.2-BA.5, and pCAGGS-XBB.1.5-EG.5-BA.2.86 constructed above were transfected into Expi293F TM Cells, collect the supernatant after 5 days, centrifuge to remove the precipitate, and then filter through a 0.22μm filter membrane to further remove impurities. The filtered cell supernatant was purified by nickel affinity column chromatography; specifically, at 4°C, the cell supernatant was passed through a nickel affinity column (Histrap, GE Healthcare), washed with buffer A (20mMTris, 150mM NaCl, pH 8.0) to remove non-specific binding proteins; then, the impurities were eluted with a low concentration of imidazole (20mM Tris, 150mM NaCl, pH 8.0, 20mM imidazole), and the target protein was eluted from HisTrap with buffer B (20mM Tris, 150mM NaCl, pH 8.0, 300mM imidazole), and the eluate was concentrated more than 30 times with a 10kDa concentrator to replace the buffer A, with a final volume of less than 1mL; finally, Superdex TMThe target protein was further purified by molecular sieve chromatography using a 200 Increase 10 / 300 GL column (GE Healthcare). The molecular sieve chromatography buffer was PBS buffer (8 mM Na2HPO4, 136 mM NaCl, 2 mM KH2PO4, 2.6 mM KCl, pH 7.4).

[0120] The molecular sieve chromatography curves of the recombinant trimer antigens PPP, D25 and XE6 and the SDS-PAGE identification results of the eluates at their elution peaks are shown in the figure below: Figure 1 , Figure 2 , Figure 3 shown by Figure 1 , 2 3. It can be seen that PPP, D25 and XE6 all have an elution peak at an elution volume of about 13.5 mL. SDS-PAGE analysis of the eluate at the elution peak shows that under non-reducing (loading buffer without dithiothreitol DTT) and reducing (loading buffer containing dithiothreitol DTT) conditions, the eluted protein size is about 75 KDa, which is consistent with the molecular size of the above two trimeric proteins. This shows that the three recombinant antigen proteins are all stable in the form of disulfide-independent trimers, and that PPP, D25 and XE6 RBD trimer proteins have indeed been purified, and the electrophoresis band is single, indicating that the purified protein has a high purity. In addition, PPP, D25 and XE6 RBD trimer proteins also have a high yield.

[0121] Example 3: Experimental animal immunization and sample collection

[0122] In order to compare the immunogenicity of the recombinant trimeric antigens PPP, D25 and XE6, the purified trimeric antigen proteins PPP, D25 or XE6 obtained in Example 2 were used as immunogens to immunize BALB / c mice, and the negative control (Sham group) was immunized with PBS solution, with 8 mice in each group. The BALB / c mice used were purchased from Vital River Company, all female, 6-8 weeks old. The mouse groups and immunization doses are shown in Table 1.

[0123] Table 1. Grouping and immunization dose of mice immunized with the novel coronavirus RBD trimer vaccine

[0124] Grouping Immunogen Dosage (μg / mouse) pcs / group Adjuvant 1 PBS / 8 Addavax 2 PPP(Prototype-Protoype-Prototype) 2 8 Addavax 3 D25 (Delta-BA.2-BA.5) 2 8 Addavax 4 XE6(XBB.1.5-EG.5-BA.2.86) 2 8 Addavax

[0125] The specific experimental procedures are as follows:

[0126] The immunogens PPP, D25 and XE6 were diluted to 40 μg / mL with PBS, and the diluted immunogens were mixed and emulsified with Addavax adjuvant at a volume ratio of 1:1 to prepare the vaccine. The negative control group was a mixture of PBS solution and Addavax adjuvant.

[0127] Immunization regimen Figure 4 As shown. Specifically, the vaccine obtained according to the above method was used to immunize BALB / c mice by leg muscle injection. All mice were immunized for the first, second and third time on day 0, day 21 and day 42, respectively, with an inoculation volume of 100 μL each time (containing 2 μg of antigen protein), 50 μL per leg. On days 19, 35 and 56, blood was collected from the orbital venous plexus of the mice, and serum was collected by centrifugation. The obtained serum was inactivated at 56°C for 30 minutes and stored in a -80°C refrigerator for determination of antigen-specific antibody titers and pseudovirus neutralizing antibody titers.

[0128] Example 4: Detection of antigen-specific antibody titers induced by recombinant trimer antigen protein vaccine by enzyme-linked immunosorbent assay (ELISA)

[0129] In this example, the antigen-specific antibody titer of the serum of mice immunized with the recombinant trimeric antigen protein vaccines PPP, D25 and XE6 in Example 3 was detected by enzyme-linked immunosorbent assay (ELISA), and the specific procedure was as follows:

[0130] (1) Dilute PPP, D25 and XE6 trimer antigen proteins to 3 μg / mL using ELISA coating solution (Solebol, C1050), add 100 μL of the dilution solution to each well of a 96-well ELISA plate (Coring, 3590), and place at 4°C overnight (more than 12 h) for coating;

[0131] (2) Pour off the coating solution, add PBS, and wash once; use 5% skim milk prepared in PBS as blocking solution, add 100 μL per well to a 96-well plate, leave at room temperature for 1 hour for blocking, and then wash once with PBS solution;

[0132] (3) During the blocking period, the mouse serum samples were diluted with blocking solution; the serum samples were diluted in a 4-fold gradient starting from 20 times; specifically, 152 μL of blocking solution and 8 μL of serum sample were added to the first well and mixed evenly, and the second dilution was 120 μL of blocking solution and 40 μL of the solution in the first well, and then diluted in sequence; after dilution, 100 μL was added to each well of the ELISA plate, and the blocking solution was added to the negative control group, incubated at 37°C for 2 hours, and then washed 4 times with PBS-T;

[0133] (4) Add HRP-conjugated goat anti-mouse secondary antibody (Bio-Edge, BE0102-100) diluted 1:2000 with blocking solution to each well, incubate at 37°C for 1.5 h, and then wash three times with PBST; then, add 60 μL of TMB color development solution for color development. After an appropriate reaction time, add 60 μL of 2 M hydrochloric acid to terminate the reaction, and measure the OD450 value on an enzyme reader.

[0134] The antibody titer value is defined as the highest serum dilution factor with a reaction value greater than 2.5 times the negative control value. When the reaction value of the lowest dilution factor (detection limit) is still less than 2.5 times the background value, the titer of the sample is defined as half of the lowest dilution factor, i.e. 1:10.

[0135] The immunogenicity test results of the serum collected on day 19 (i.e., one immune serum) are as follows Figure 5 As shown; Figure 5 The results showed that PPP, D25 and XE6 trimer vaccines all produced corresponding binding antibodies after immunization, and the specific binding antibody titers of the serum after immunization with the three immunogens were all above 10 3 ~10 4 There was no significant difference among the three immunization groups.

[0136] The immunogenicity test results of the serum collected on day 35 (i.e., the second immunization serum) are as follows Figure 6 As shown: Figure 6 The results showed that the binding antibody titers induced by PPP, D25 and XE6 trimer vaccines in mice after the second immunization were all above 10 6 There was no significant difference among the three immunization groups.

[0137] The immunogenicity test results of the serum collected on day 56 (i.e., triple-immune serum) are as follows: Figure 7 As shown: Figure 7 The results showed that the antibody titers of the PPP, D25 and XE6 trimer vaccine groups were all above 10 6 ~10 7 There was no significant difference among the three immunization groups.

[0138] The above experimental results show that the three immunogens, PPP, D25 and XE6, can induce mice to produce high titers of antigen-specific binding antibodies. After the third immunization, the binding antibody titer can reach 10 6 -10 7 .

[0139] Example 5: Detecting the neutralizing antibody titer produced by the recombinant trimer antigen protein vaccine against the new coronavirus pseudovirus through pseudovirus neutralization experiment

[0140] Using the new coronavirus pseudovirus, the 50% pseudovirus neutralization titer (pVNT) of the three immune sera (i.e., serum collected on the 56th day) of mice immunized with the recombinant trimeric antigen protein vaccines PPP, D25 and XE6 in Example 3 was tested against the prototype strain of the new coronavirus, the Delta variant, and pseudoviruses of Omicron BA.1, BA.2, BA.5, XBB, BA.2.86, EG.5, JN.1 and KP.2 subtypes. 50 ).

[0141] The new coronavirus pseudovirus used in this example is a pseudovirus displaying the new coronavirus S protein prepared based on the vesicular stomatitis virus (VSV) backbone. For the preparation method, please refer to the method section of the paper that this research group has published publicly (Effects of a Prolonged Booster Interval on Neutralization of Omicron Variant, N Engl J Med, 2022, PMID: 35081296).

[0142] The method for detecting the neutralizing antibody titer of the new coronavirus pseudovirus (hereinafter referred to as pseudovirus) is as follows:

[0143] In a 96-well plate, the immune mouse serum was diluted in a 2-fold gradient with an initial concentration of 1:20, and a total of 11-14 concentration gradients were set; then, the diluted immune mouse serum was mixed with the pseudovirus (blank culture medium mixed with pseudovirus as a negative control (NC), and blank culture medium not mixed with pseudovirus as a blank control (MOCK), and incubated at 37°C for 1 hour; then, the immune mouse serum-pseudovirus mixture was transferred to a 96-well plate filled with Vero cells, and after incubation at 37°C for 15 hours, the positive cell value was detected and calculated by a CQ1 confocal cell imager (Yokogawa), and then, a fitting curve was drawn in the GraphPadPrism software to calculate the reciprocal of the serum dilution corresponding to 50% neutralization, which was the 50% pseudovirus neutralization titer pVNT. 50 .

[0144] The results of the neutralizing antibody titers produced by the mice in each immunization group in the serum after three immunizations against the prototype strain, Delta and Omicron BA.1, BA.2, BA.5, XBB, EG.5, BA.2.86, JN.1 and KP.2 subtype pseudoviruses are shown as follows: Figure 8-17 shown.

[0145] Depend on Figure 8-17 The experimental results shown are as follows:

[0146] 1) If Figure 8Shown: pVNT of serum from mice in the PPP immunization group against the pseudovirus of the prototype strain of the new coronavirus 50 pVNT of serum from mice immunized at 41,141,D25 50 18,858, pVNT of serum from mice immunized with XE6 50 It is 45. Statistical analysis showed that for the prototype pseudovirus, the PPP immunization group was significantly higher than the D25 immunization group (*) and the XE6 immunization group (***), and the D25 immunization group was significantly higher than the XE6 immunization group (***).

[0147] 2) If Fig. 9 Shown: Pseudovirus of the Delta variant of the new coronavirus, pVNT of the serum of mice in the PPP immunization group 50 14,274, pVNT of serum from mice immunized on D25 50 25,400, pVNT of serum from mice immunized with XE6 50 It is 36. Statistical analysis showed that for the delta variant pseudovirus, there was no significant difference between the PPP immunization group and the D25 immunization group, the PPP immunization group was significantly higher than the XE6 immunization group (***), and the D25 immunization group was significantly higher than the XE6 immunization group (***).

[0148] 3) If Fig.10 Shown: Pseudovirus of SARS-CoV-2 Omicron BA.1 variant, pVNT of PPP-immunized mouse serum 50 3,154, pVNT of serum from mice immunized on D25 50 37,739, pVNT of serum from mice immunized with XE6 50 It is 273. Statistical analysis showed that for the Omicron BA.1 variant pseudovirus, the D25 immunization group was significantly higher than the PPP immunization group (**) and the XE6 immunization group (**), and the PPP immunization group was significantly higher than the XE6 immunization group (*).

[0149] 4) If Fig.11 Shown: Pseudovirus of PPP-immunized mice serum against the novel coronavirus Omicron BA.2 variant 50 5,558, pVNT of serum from mice immunized on D25 50 The pVNT of serum from mice immunized with XE6 was 42,588. 50 The number was 1,553. Statistical analysis showed that for the Omicron BA.2 variant pseudovirus, the D25 immunization group was significantly higher than the PPP immunization group (**) and the XE6 immunization group (***), and there was no significant difference between the PPP immunization group and the XE6 immunization group.

[0150] 5) If Fig.12 Shown: pVNT of serum from mice in the PPP immunization group against pseudovirus of the novel coronavirus Omicron BA.5 variant 50 1,362, pVNT of serum from mice immunized on D25 50 25,399, pVNT of serum from mice immunized with XE6 50 The number was 4,736. Statistical analysis showed that for the Omicron BA.5 variant pseudovirus, the D25 immunization group was significantly higher than the PPP immunization group (**) and the XE6 immunization group (*), and there was no significant difference between the PPP immunization group and the XE6 immunization group.

[0151] 6) If Fig.13 Shown: Pseudovirus of SARS-CoV-2 Omicron XBB variant, pVNT of PPP-immunized mouse serum 50 pVNT of serum from mice immunized on D25 50 pVNT of serum from mice immunized with 556 and XE6 50 The number was 2,733. Statistical analysis showed that for the Omicron XBB variant pseudovirus, the XE6 immunization group was significantly higher than the PPP immunization group (***) and the D25 immunization group (**), and the D25 immunization group was significantly higher than the PPP immunization group (***).

[0152] 7) If Fig.14 Shown: Pseudovirus of novel coronavirus Omicron EG.5 variant, pVNT of PPP immunized mouse serum 50 pVNT of serum from mice immunized on D25 50 pVNT of serum from mice immunized with 586 and XE6 50 The number was 7,353. Statistical analysis showed that for the Omicron EG.5 variant pseudovirus, the D25 immunization group (**) and the XE6 immunization group (***) were significantly higher than the PPP immunization group, and the XE6 immunization group was significantly higher than the D25 immunization group (***).

[0153] 8) If Fig.15 Shown: Pseudovirus of novel coronavirus Omicron BA.2.86 variant, pVNT of PPP immunized mouse serum 50 pVNT of serum from mice immunized on D25 50 pVNT of 464,XE6 immunized mouse serum 50 The number was 2,289. Statistical analysis showed that for the Omicron XBB variant pseudovirus, the XE6 immunization group (***) and the D25 immunization group (***) were significantly higher than the PPP immunization group, and there was no significant difference between the XE6 immunization group and the D25 immunization group.

[0154] 9) If Fig.16 Shown: Pseudovirus against the novel coronavirus Omicron JN.1 variant, pVNT of PPP-immunized mouse serum 50 pVNT of serum from mice immunized on D25 50 pVNT of serum from mice immunized with 209 and XE6 50 The number was 1,484. Statistical analysis showed that for the Omicron JN.1 variant pseudovirus, the XE6 immunization group (***) and the D25 immunization group (***) were significantly higher than the PPP immunization group, and the XE6 immunization group was significantly higher than the D25 immunization group (***).

[0155] 10) As Fig.17 Shown: Pseudovirus of SARS-CoV-2 Omicron KP.2 variant, pVNT of PPP-immunized mouse serum 50 pVNT of serum from mice immunized on D25 50 pVNT of serum from mice immunized with 229 and XE6 50 The number was 1,359. Statistical analysis showed that for the Omicron KP.2 variant pseudovirus, the XE6 immunization group (***) and the D25 immunization group (***) were significantly higher than the PPP immunization group, and the XE6 immunization group was significantly higher than the D25 immunization group (**).

[0156] The radar chart of the neutralizing antibody titer test results against different strains of pseudovirus produced in the serum of mice in each immunization group after three immunizations is shown in the figure below. Fig.18 As shown; Fig.18 show:

[0157] (1) Compared with the PPP immunization group, the sera of mice immunized with D25 showed higher neutralizing protective activity against Delta, Omicron BA.1, BA.2, BA.5, XBB, EG.5, BA.2.86, JN.1, and KP.2 variants;

[0158] In particular, for XBB, EG.5, BA.2.86, JN.1 and KP.2, the serum of mice in the PPP immunization group has basically lost its pseudovirus neutralizing activity against these new coronavirus mutant strains; while the serum of mice in the D25 group has a strong pseudovirus neutralizing titer against the prototype strain, Delta, Omicron BA.1, BA.2 and BA.5, and it still has a certain neutralizing protection activity (greater than 100) against the later-appearing XBB, EG.5, BA.2.86, JN.1 and KP.2.

[0159] In other words, the D25 trimer vaccine is significantly higher than PPP in terms of neutralizing effect against pseudoviruses of the new coronavirus prototype strain, Delta, Omicron BA.1, BA.2, BA.5, XBB, EG.5, BA.2.86, JN.1 and KP.2 mutant strains.

[0160] (2) Compared with the D25 immunization group, the sera of mice in the XE6 immunization group showed higher neutralizing protective activity against Omicron XBB, EG.5, BA.2.86, JN.1, and KP.2 variants;

[0161] As the later strains of the new coronavirus mutated, the neutralization protection titer of D25 gradually decreased, especially the neutralization protection for Omicron XBB, EG.5, BA.2.86, JN.1 and KP.2; in contrast, XE6 produced a high level of neutralization titer in a balanced manner for various types of Omicron variants (including BA.1, BA.2, BA.5, XBB, EG.5, BA.2.86, JN.1 and KP.2), and its neutralization titer did not decrease significantly as the new coronavirus strains mutated.

[0162] The above experiments confirmed that the XE6 trimer antigen of the present invention can induce a strong immune response against the later-appearing Omicron BA.1, BA.2, BA.5, XBB, EG.5, BA.2.86, JN.1 and KP.2 mutant strains, indicating that it will have a good immune protection effect against a variety of new coronavirus Omicron mutant strains and has broad application prospects.

[0163] Example 6: Preparation of XE6 mRNA vaccine

[0164] The preparation process of XE6 mRNA vaccine is as follows:

[0165] (1) Construction of pcDNA3.1 expression plasmid of XBB.1.5-EG.5-BA.2.86

[0166] The coding region of the recombinant expression plasmid pCAGGS-XBB.1.5-EG.5-BA.2.86 constructed in Example 2 was cloned into the pcDNA 3.1 vector with 5'UTR, 3'UTR and Ploy A tail added to construct the pcDNA3.1 expression plasmid of XBB.1.5-EG.5-BA.2.86, and the correctness of the construction was verified by sequencing.

[0167] (2) The correctly sequenced plasmid is cultured and extracted.

[0168] (3) Plasmid linearization

[0169] The enzyme digestion was carried out at 37℃ for 4h, and then inactivated at 65℃ for 20min. The digestion products were purified and recovered using a gel recovery kit. The composition of 100μl digestion system is shown in the following table:

[0170]

[0171] (4) In vitro transcription

[0172] In vitro transcription was performed using an in vitro transcription kit and then purified by lithium chloride precipitation. The specific process is as follows:

[0173] 1) Mix all components except T7 RNA Polymerase Mix by vortexing, centrifuge briefly, collect the residue at the bottom of the tube, and store on ice for later use.

[0174] 2) Prepare the in vitro transcription system according to the following table:

[0175]

[0176] 3) Mix the components gently with a pipette, collect by brief centrifugation, and incubate at 37°C for 4 h.

[0177] 4) Product RNA is purified by lithium chloride

[0178] a. Add 1 / 2 volume of Lithium Chloride Precipitation Solution to the system.

[0179] b. Mix well and place in -20℃ for 30 min.

[0180] c. Centrifuge at 12000 rpm for 15 min, remove the supernatant and collect the precipitate.

[0181] d. Wash three times with pre-cooled 75% ethanol.

[0182] e. Detect the concentration after reconstitution in RNase-free water.

[0183] (5) mRNA capping

[0184] Use the capping kit to cap the mRNA 5' end. The specific process is as follows:

[0185] 1) Dilute an appropriate amount (50 μg) of RNA to 67 μL with RNase-free water, which is a concentration of 746 ng / μL;

[0186] 2) Heat the RNA at 65°C for 10 min and immediately place on ice for 5 min;

[0187] 3) Prepare the reaction system according to the table below and place it at 37°C for 1 hour;

[0188]

[0189]

[0190] After capping, the product RNA was purified by lithium chloride, and the specific process was as follows:

[0191] a. Add 1 / 2 volume of Lithium Chloride Precipitation Solution to the system;

[0192] b. After mixing, place in -20℃ for at least 30 minutes;

[0193] c. Centrifuge at 12000 rpm for 15 min, remove the supernatant and collect the precipitate;

[0194] d. Wash three times with pre-cooled 75% ethanol;

[0195] e. Add RNase-free water to reconstitute and test the concentration

[0196] (6) mRNA packaging

[0197] 1) Prepare the liposome mixture according to the following table:

[0198]

[0199] The packaging configuration is based on 200 μg of RNA per time, and it can be diluted to 2 mL with pH 4.3 sodium acetate solution before packaging on the chip.

[0200] 2) Liposome packaging

[0201] Use three 10mL syringes with needles to draw 6mL of anhydrous ethanol (filtered), 6mL of DEPC water, and 6mL of pH4.3 sodium acetate buffer respectively. Set the syringe to 10mL in the software. First, use anhydrous ethanol (filtered) and DEPC water at a ratio of 1:1 (total flow rate 12mL / min, total volume 2mL) to clean the chip. Then replace the DEPC water with pH4.3 sodium acetate buffer and clean the chip again at a ratio of 1:1 (total flow rate 12mL / min, total volume 2mL).

[0202] 150 μg mRNA was diluted to 1.5 mL with pH 4.3 sodium acetate buffer, and the liposome mixture was drawn with a 5 mL screw-on syringe without a needle + a 5 mL syringe needle, and anhydrous ethanol (filtered) was replaced. The diluted mRNA was drawn with a 3 mL screw-on syringe without a needle + a 2 mL syringe needle, and the pH 4.3 sodium acetate buffer was replaced. The syringe specifications were set to 3 mL and 5 mL, respectively, and packaged at a ratio of 3:1 (total flow rate 12 mL / min, total volume 1.9 mL, waste 0.1 mL); after packaging, the chip was first cleaned with anhydrous ethanol (filtered) and pH 4.3 sodium acetate buffer at a ratio of 1:1 (total flow rate 12 mL / min, total volume 2 mL), and then the pH 4.3 Replace the sodium acetate buffer with DEPC water and clean the chip again at a ratio of 1:1 (total flow rate 12 mL / min, total volume 6 mL). Use two empty 10 mL syringes to inhale air and drain the chip at a ratio of 1:1 (total flow rate 12 mL / min, total volume 6 mL).

[0203] After packaging, add 1×D-PBS to 14 mL, concentrate to 1 mL in a 10kDa concentrator, add 14 mL 1×D-PBS to replace the liquid, repeat the liquid replacement process twice, 2700 rpm, about 40 min / time, concentrate to 500 μL for the last time, and transfer to a 1.5 mL EP tube.

[0204] 3) Liposome concentration detection

[0205] Add the test sample to the brown 96-well plate, stain with the kit to detect the concentration, dilute the RNA standard 50 times (2μg / mL), dilute 20×TE to 1×TE with DEPC water, dilute the sample 100 times, add two wells to each 200 times, add Triton to permeabilize one well for each dilution concentration, and do not add Triton to the other well to detect free mRNA, dilute the colorimetric solution 200 times with 1×TE, add 100μL of colorimetric solution to each well, react at room temperature for 5 minutes, read at 480nm / 520nm, and calculate the concentration of the packaged liposomes.

[0206] After testing, the obtained XE6 mRNA vaccine meets the relevant standards for mRNA vaccines.

[0207] Example 7: Experimental Animal Immunization and Sample Collection (Comparison of Protein Vaccine and mRNA Vaccine)

[0208] In order to detect the immunogenicity of the recombinant trimer antigen XE6 protein vaccine and mRNA vaccine of the present invention and compare the two, the XE protein vaccine prepared in Example 3 and the XE6 mRNA vaccine prepared in Example 6 were used as immunogens to immunize 6-8 week old female BALB / c mice, and the negative control (Sham group) was immunized with PBS solution, with 5 mice in each group. The BALB / c mice used were purchased from Vital River Company. The mouse grouping and immunization dose are shown in Table 2. The process of immunizing mice and collecting samples is shown in Figure 4 shown.

[0209] Table 2 Comparison of the efficacy of the novel coronavirus RBD trimer vaccine Grouping and immunization dose of immunized mice

[0210]

[0211] Example 8: Detection of antigen-specific antibody titers induced by XE6 protein vaccine and mRNA vaccine by enzyme-linked immunosorbent assay (ELISA)

[0212] The ELISA test was performed according to the experimental method of Example 4.

[0213] The results of the RBD-specific binding antibody titers of the first, second, and third immunization sera of mice immunized with XE6 protein vaccine and mRNA vaccine are shown in Figure 2. Fig.19 , 20 , as shown in 21; Fig.19 , 20 , 21 shows:

[0214] (1) The immunogenicity test results of the serum collected on day 19 (i.e., the first immune serum) are as follows Fig.19 As shown; Fig.19 The results showed that XE6 protein 1μg, XE6 protein 5μg, XE6 mRNA 1μg, and XE6 mRNA 5μg trimer vaccines all produced corresponding antibodies after immunization, and the combined antibody titer was between 10 2 -10 4 The specific binding antibody titer of serum after one vaccination with 1 μg of XE6 protein trimer vaccine was about 10 2 , while the antibody titer of the XE6 protein 5μg immunization group was close to 10 3 That is, the specific binding antibody titer of the XE6 protein 5μg group was significantly higher than that of the XE6 protein 1μg group (*); while the specific binding antibody titer of the XE6 mRNA group was significantly higher than that of the XE6 protein group with the same dose (**), but the difference between the XE6 mRNA 1μg group and the XE6 mRNA 5μg group was not significant, and the antibody titer values ​​were between 10 3 -10 4 ;

[0215] (2) The immunogenicity test results of the serum collected on day 35 (i.e., the second immunization serum) are as follows: Fig. 20 As shown: Fig. 20 The results showed that the XE6 protein 1μg, XE6 protein 5μg, XE6 mRNA 1μg, and XE6 mRNA 5μg trimer vaccines all produced corresponding antibodies after immunization, and the combined antibody titer was between 10 5 -10 7 The specific binding antibody titer of serum after one vaccination with 1μg and 5μg of XE6 protein trimer vaccine was close to 10 6 , and the difference between the two groups was not significant; the binding antibody titer of the XE6 mRNA 1μg immunization group was significantly higher than that of the XE6 protein 1μg group (**); the binding antibody titer of the XE6 mRNA 5μg immunization group was significantly higher than that of the XE6 protein 5μg group (*); the difference between the XE6 mRNA 1μg group and the XE6 mRNA 5μg group was not significant, and the antibody titer values ​​were between 10 6 -10 7 .

[0216] (3) The immunogenicity test results of the serum collected on day 56 (i.e., triple-immune serum) are as follows: Fig.21 As shown: Fig.21 The results showed that XE6 protein 1μg, XE6 protein 5μg, XE6 mRNA 1μg, and XE6 mRNA 5μg trimer vaccines all produced high titers of specific binding antibodies after immunization. 5 -10 8 The specific binding antibody titer of serum after one vaccination with 1μg and 5μg of XE6 protein trimer vaccine was close to 10 6 , and the XE6 protein 5μg group was significantly higher than the 1μg group (*); the XE6 mRNA 1μg immunization group had a significantly higher binding antibody titer than the XE6 protein 1μg group (**); the XE6 mRNA 5μg immunization group had a significantly higher binding antibody titer than the XE6 protein 5μg group (**); the difference between the XE6 mRNA 1μg group and the XE6 mRNA 5μg group was not significant, and the antibody titer values ​​were close to 10 7 .

[0217] The above results show that compared with the XE6 protein 1μg group, the XE6 protein 5μg group induced higher antigen-specific binding antibody titers; compared with the XE6 protein vaccine, the same dose of XE6 mRNA vaccine induced higher antigen-specific binding antibody titers. This shows that the XE6 mRNA vaccine may have a higher immune protection effect than the XE6 protein vaccine.

[0218] Example 9: Neutralizing antibody titers produced by the serum of mice immunized with XE6 protein vaccine and mRNA vaccine against pseudoviruses of various novel coronavirus strains were detected by pseudovirus neutralization experiments

[0219] The pseudovirus neutralization experiment was performed according to the experimental method of Example 5.

[0220] The neutralization results of the secondary sera of mice immunized with XE6 protein vaccine and mRNA vaccine against pseudoviruses of Omicron XBB.1.5, Omicron EG.5, and Omicron BA.2.86 are shown in the table below. Fig. 22 , 23 , as shown in 24; Fig. 22 , 23 ,24Display:

[0221] (1) The results of serum collected on day 35 (i.e., second-immune serum) neutralizing the pseudovirus against Omicron XBB.1.5 are as follows Fig.24 As shown: XE6 protein 1μg group (248), XE6 protein 5μg group (482), XE6 mRNA 1μg group (6,877), XE6 mRNA 5μg group (22,296). There was no significant difference between the XE6 protein 1μg group and the XE6 protein 5μg group; the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 mRNA 1μg group (**); the neutralization titer of the XE6 mRNA 1μg group was significantly higher than that of the XE6 protein 1μg group (**); the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 protein 1μg group (**).

[0222] (2) The results of serum collected on day 35 (i.e., second-immune serum) neutralizing the pseudovirus against Omicron EG.5 are as follows: Fig.25 As shown: XE6 protein 1μg group (226), XE6 protein 5μg group (197), XE6 mRNA 1μg group (6,746), XE6 mRNA 5μg group (19,615). There was no significant difference between the XE6 protein 1μg group and the XE6 protein 5μg group; the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 mRNA 1μg group (**); the neutralization titer of the XE6 mRNA 1μg group was significantly higher than that of the XE6 protein 1μg group (**); the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 protein 1μg group (**).

[0223] (3) The results of serum collected on day 35 (i.e., second-immune serum) neutralizing the pseudovirus against Omicron BA.2.86 are as follows: Fig.26As shown: XE6 protein 1μg group (93), XE6 protein 5μg group (15), XE6 mRNA 1μg group (4,602), XE6 mRNA 5μg group (22,208). The XE6 protein 1μg group was significantly higher than the XE6 protein 5μg group, but the titers were very low; the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 mRNA 1μg group (**); the neutralization titer of the XE6 mRNA 1μg group was significantly higher than that of the XE6 protein 1μg group (**); the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 protein 1μg group (**).

[0224] The radar chart of the neutralization results of the second immune serum against the pseudoviruses of Omicron XBB.1.5, Omicron EG.5, and Omicron BA.2.86 is shown in Fig.25 , Fig.25 The results showed that for XBB.1.5, EG.5 and BA.2.86, the XE6 mRNA 5μg immunization group was significantly better than the XE6 protein 5μg immunization group; the XE6 mRNA 1μg immunization group was significantly better than the XE6 protein 1μg immunization group; the XE6 mRNA 5μg immunization group was significantly better than the XE6 mRNA 1μg immunization group; the difference between the XE6 protein 5μg immunization group and the XE6 protein 1μg immunization group was not very significant.

[0225] The results of the three immune sera of mice immunized with XE6 protein vaccine and mRNA vaccine against pseudoviruses of Omicron XBB.1.5, Omicron EG.5, and Omicron BA.2.86 are shown as follows: Fig.26 , 27 , as shown in 28:

[0226] (1) The results of serum collected on day 56 (i.e., second-immune serum) neutralizing the pseudovirus against Omicron XBB.1.5 are as follows Fig.26 As shown: XE6 protein 1μg group (5,797), XE6 protein 5μg group (7,649), XE6 mRNA 1μg group (19,431), XE6 mRNA 5μg group (206,373). There was no significant difference between the XE6 protein 1μg group and the XE6 protein 5μg group; the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 mRNA 1μg group (***); the neutralization titer of the XE6 mRNA 1μg group was significantly higher than that of the XE6 protein 1μg group (**); the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 protein 1μg group (***).

[0227] (2) The results of serum collected on day 56 (i.e., second-immune serum) neutralizing the pseudovirus against Omicron EG.5 are as follows: Fig. 27 As shown: XE6 protein 1μg group (6,384), XE6 protein 5μg group (11,581), XE6 mRNA 1μg group (23,781), XE6 mRNA 5μg group (218,882). There was no significant difference between the XE6 protein 1μg group and the XE6 protein 5μg group; the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 mRNA 1μg group (***); the neutralization titer of the XE6 mRNA 1μg group was significantly higher than that of the XE6 protein 1μg group (**); the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 protein 1μg group (***).

[0228] (3) The results of serum collected on day 56 (i.e., second-immune serum) neutralizing the pseudovirus against Omicron BA.2.86 are as follows: Fig.28 As shown: XE6 protein 1μg group (93), XE6 protein 5μg group (15), XE6 mRNA 1μg group (4,602), XE6 mRNA 5μg group (22,208). There was no significant difference between the XE6 protein 1μg group and the XE6 protein 5μg group; the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 mRNA 1μg group (**); the neutralization titer of the XE6 mRNA 1μg group was significantly higher than that of the XE6 protein 1μg group (**); the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 protein 1μg group (**).

[0229] The radar chart of the neutralization results of the three immune sera against the pseudoviruses of Omicron XBB.1.5, Omicron EG.5, and Omicron BA.2.86 is shown in Fig.29 , Fig.29 The results showed that for XBB.1.5, EG.5 and BA.2.86, the XE6 mRNA 5μg immunization group was significantly better than the XE6 protein 5μg immunization group; the XE6 mRNA 1μg immunization group was significantly better than the XE6 protein 1μg immunization group; the XE6 mRNA 5μg immunization group was significantly better than the XE6 mRNA 1μg immunization group; the difference between the XE6 protein 5μg immunization group and the XE6 protein 1μg immunization group was not very significant.

[0230] In short, for the evaluation of the neutralization effects of three new coronavirus pseudoviruses, Omicron XBB.1.5, EG.5 and BA.2.86, there was no significant difference between the XE6 protein 1μg group and the XE6 protein 5μg group; the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 mRNA 1μg group; the neutralization titer of the XE6 mRNA 1μg group was significantly higher than that of the XE6 protein 1μg group; the neutralization titer of the XE6 mRNA 5μg group was significantly higher than that of the XE6 protein 1μg group.

[0231] In summary, the recombinant chimeric antigen XE6 of the present invention can induce high levels of binding antibodies and neutralizing antibodies against various types of Omicron variants (including BA.1, BA.2, BA.5, XBB, EG.5, BA.2.86, JN.1 and KP.2), and its neutralizing titer for subsequent new coronavirus variants has not changed significantly, so it can cope with the current and future complex new coronavirus epidemics. In addition, compared with the mRNA vaccine and protein vaccine of the recombinant chimeric antigen XE6 of the present invention, the mRNA vaccine can induce mice to produce higher antigen-specific binding antibody titers and new coronavirus pseudovirus neutralizing antibody titers, and has the potential to become a candidate for a new generation of new coronavirus vaccines. The new coronavirus strains change rapidly, and the types and immunological characteristics of future epidemic strains are extremely difficult to predict. Therefore, the above characteristics of the XE6 vaccine have great application value for preventing changes in epidemic strains or the co-epidemic of multiple strains.

[0232] 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.

[0233] The sequences used in this article are as follows:

[0234] SEQ ID NO:1 - Amino acid sequence of the R319-V534 region of the S protein RBD of the Omicron XBB.1.5 variant (216aa)

[0235] RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLP DDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLV

[0236] SEQ ID NO:2 – Amino acid sequence of the V320-V534 region of the S protein RBD of the Omicron EG.5 variant (215aa)

[0237] VQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLLRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLV

[0238] SEQ ID NO:3 - Amino acid sequence of the V320-K537 region of the S protein RBD of the Omicron BA.2.86 variant (217aa)

[0239] VQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRLFRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0240] SEQ ID NO:4—XE6 antigen without signal peptide and His tag (648aa)

[0241] RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLLRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVVQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSFRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0242] SEQ ID NO:5—Signal peptide (17aa)

[0243] MIHSVFLLMFLLTPTES

[0244] SEQ ID NO:6—XE6 antigen with signal peptide and His tag (671 aa)

[0245] MIHSVFLLMFLLTPTESRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLLRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVVQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSFRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKHHHHHH*

[0246] SEQ ID NO:7 - DNA encoding SEQ ID NO:4 (1944 bp)

[0247]

[0248] SEQ ID NO:8 - DNA encoding SEQ ID NO:6 (2013 bp)

[0249]

[0250] SEQ ID NO:9 - mRNA encoding SEQ ID NO:4 (1944 bp)

[0251]

[0252] SEQ ID NO:10 - mRNA encoding SEQ ID NO:6 (2013 bp)

[0253]

[0254] SEQ ID NO: 11 - DNA sequence encoding PPP construct (2040 bp)

[0255]

[0256] SEQ ID NO: 12—DNA sequence encoding D25 construct (2016 bp)

[0257]

Claims

1. A recombinant chimeric antigen of a novel coronavirus, characterized in that: The recombinant chimeric antigen has an amino acid sequence as shown in formula (I): (AB)-C1-(A-B')-C2-(AB”) (I) In formula (I): AB represents the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Omicron variant XBB.1.5 subtype or a portion 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; A-B' represents the amino acid sequence of the RBD domain of the S protein of the EG.5 subtype of the novel coronavirus Omicron variant or a portion thereof, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and has the same or substantially the same immunogenicity thereto; "AB" means the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Omicron variant BA.2.86 subtype or a portion 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; C1 and C2 are the same or different, and each independently represents a linker (GGS) n ; Wherein, n=0,1,2,3,4 or 5.

2. The recombinant chimeric antigen according to claim 1, characterized in that A portion of the RBD domain of the S protein of the novel coronavirus Omicron variant XBB.1.5 subtype is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of its entire amino acid sequence; And / or, a portion of the RBD domain of the S protein of the novel coronavirus Omicron variant EG.5 subtype is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of its entire amino acid sequence; And / or, a portion of the RBD domain of the S protein of the novel coronavirus Omicron variant BA.2.86 subtype is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of its entire amino acid sequence; And / or, n=0, 1, 2 or 3.

3. The recombinant chimeric antigen according to claim 1 or 2, characterized in that: The amino acid sequence of the RBD domain of the S protein of the novel coronavirus Omicron variant XBB.1.5 subtype or a portion thereof is as shown in SEQ ID NO: 1, or an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1, and having the same or substantially the same immunogenicity as the amino acid sequence; And / or, the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Omicron variant EG.5 subtype or a portion thereof is as shown in SEQ ID NO: 2, or an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2, and having the same or substantially the same immunogenicity as that thereof; And / or, the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Omicron variant BA.2.86 subtype or a portion thereof is as shown in SEQ ID NO: 3, or an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 3, and having the same or substantially the same immunogenicity as that thereof; And / or, n=0, 1 or 2.

4. The recombinant chimeric antigen according to any one of claims 1 to 3, characterized in that: AB represents the amino acid sequence shown in SEQ ID NO: 1, A-B' represents the amino acid sequence shown in SEQ ID NO: 2, and AB" represents the amino acid sequence shown in SEQ ID NO: 3; Preferably, the recombinant chimeric antigen comprises the amino acid sequence shown in SEQ ID NO:

4.

5. The recombinant chimeric antigen according to claim 4, characterized in that The N-terminus of the recombinant chimeric antigen further includes a signal peptide sequence, preferably, the signal peptide sequence is as shown in SEQ ID NO: 5; And / or, the C-terminus of the recombinant chimeric antigen further comprises a tag sequence, preferably, the tag is a 6×His tag; More preferably, the recombinant chimeric antigen comprises the amino acid sequence shown in SEQ ID NO:

6.

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

7. The preparation method according to claim 6, characterized in that: The expression system cells are mammalian cells, insect cells, yeast cells or bacterial cells; Optionally, the mammalian cell is a HEK293T cell, a 293F series cell or a CHO cell; further optionally, the 293F series cell is a HEK293F cell, a Freestyle293F cell or an Expi293F cell; Optionally, the insect cell is a sf9 cell, a Hi5 cell, a sf21 cell or a S2 cell; Optionally, the yeast cell is a Pichia pastoris cell or a yeast cell transformed therefrom; Optionally, the bacterial cells are Escherichia coli cells.

8. A polynucleotide encoding the recombinant chimeric antigen according to any one of claims 1 to 5.

9. The polynucleotide according to claim 8, characterized in that The polynucleotide is DNA or mRNA; Preferably, the DNA comprises a DNA sequence as shown in SEQ ID NO: 7 or 8; Preferably, the mRNA comprises the DNA sequence shown in SEQ ID NO:9 or 10.

10. A nucleic acid construct comprising the polynucleotide according to claim 8 or 9, and optionally, at least one expression control element operably linked to the polynucleotide. An expression vector comprising the nucleic acid construct according to claim 10 .

12. A host cell transformed or transfected with the polynucleotide according to claim 8 or 9, the nucleic acid construct according to claim 10 or the expression vector according to claim 11.

13. Use of the recombinant chimeric antigen according to any one of claims 1 to 5, the polynucleotide according to claim 8 or 9, the nucleic acid construct according to claim 10, the expression vector according to claim 11 or the host cell according to claim 12 in the preparation of a vaccine for preventing and / or treating novel coronavirus infection; Preferably, the novel coronavirus is one or more selected from the following: SARS-CoV-2 original strain, SARS-CoV-2 variant strains Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2), Omicron subtype BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4, BA.5, XBB, EG.5, BA.2.86, JN.1, KP.2; Preferably, the vaccine is used for single immunization or sequential immunization with other types of new coronavirus vaccines; further preferably, the other types of new coronavirus vaccines are inactivated vaccines.

14. A vaccine or immunogenic composition comprising the recombinant chimeric antigen according to any one of claims 1 to 5, the polynucleotide according to claim 8 or 9, the nucleic acid construct according to claim 10, the expression vector according to claim 11 or the host cell according to claim 12, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

15. The vaccine or immunogenic composition according to claim 14, which is a new coronavirus recombinant protein vaccine, comprising a recombinant chimeric antigen according to any one of claims 1 to 5 and an adjuvant; Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant; further optionally, the adjuvant is a MF59-like adjuvant; further optionally, the adjuvant is Addavax.

16. The vaccine or immunogenic composition according to claim 14, which is a novel coronavirus DNA vaccine, comprising: (i) eukaryotic expression vector; and (ii) a DNA sequence encoding a recombinant chimeric antigen having a structure as shown in formula (I) constructed into the eukaryotic expression vector, preferably a DNA sequence as shown in SEQ ID NO: 7 or 8; Preferably, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

17. The vaccine or immunogenic composition according to claim 14, which is a novel coronavirus mRNA vaccine, comprising: (I) an mRNA sequence encoding a recombinant chimeric antigen having a structure as shown in formula (I), preferably an mRNA sequence as shown in SEQ ID NO: 9 or 10; and (II) Lipid nanoparticles.

18. The vaccine or immunogenic composition according to claim 14, which is a novel coronavirus-viral vector vaccine, comprising: (1) Viral backbone vector; and (2) a DNA sequence encoding a recombinant chimeric antigen having a structure as shown in formula (I) constructed into the viral backbone vector, preferably a DNA sequence as shown in SEQ ID NO: 7 or 8; 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.

19. The vaccine or immunogenic composition according to claim 14, which is a novel coronavirus nanoparticle vaccine; Preferably, the carrier of the nanoparticle is ferritin, and the recombinant chimeric antigen according to any one of claims 1 to 5 is covalently linked to ferritin and self-assembled into nanoparticles, so that the recombinant chimeric antigen is presented on the surface of the nanoparticle; further preferably, the recombinant chimeric antigen is connected to ferritin via a linker, or is covalently linked via a SpyTag / SpyCatcher connection system.

20. The vaccine or immunogenic composition according to any one of claims 14 to 19, characterized in that The vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository or parenteral formulation; Preferably, the nasal spray is selected from aerosols, sprays and powder sprays; Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film coatings and ointments; Further preferably, the tablet is a sublingual tablet; Further preferably, the granules are fine granules; Further preferably, the powder is a powder; Further preferably, the pills are pellets; Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid preparation, or an injectable preparation; further preferably, the injectable preparation is a pushable preparation.

21. A kit comprising a vaccine or immunogenic composition as claimed in any one of claims 14 to 20, and optionally other types of novel coronavirus vaccines, wherein the vaccine or immunogenic composition and the other types of novel coronavirus vaccines are packaged separately; Preferably, the other type of new coronavirus vaccine is an inactivated new coronavirus vaccine.

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