Sand bay coronavirus recombinant trimer immunogen and application thereof

Abstract

The invention relates to a recombinant trimer immunogen with a broad-spectrum and high-level immune protection effect on a Sabebe coronavirus and application of the recombinant trimer immunogen. The recombinant trimer immunogen is formed by recombining and chimeric S protein RBD structural domains or functional active fragments of the S protein RBD structural domains of two SARS-CoV-2 virus strains and S protein RBD structural domains or functional active fragments of the S protein RBD structural domains of SARS-CoV. The recombinant trimer immunogen not only can provide balanced and efficient immune protection for an SARS-CoV-2 prototype strain, a D164G mutant strain and various types of variants of an Omicron pedigree, but also can efficiently stimulate an organism to generate high-level neutralizing antibodies aiming at various other sabbe coronavirus viruses; according to the present invention, the Sarabis coronavirus immunogen has the broad-spectrum and high-efficiency immune protection effect on the Sarabis coronavirus, such that the Sarabis coronavirus immunogen has the great potential to become the core immunogen of the broad-spectrum vaccine for the Sarabis coronavirus, and has high clinical application value and high clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a Sabella coronavirus recombinant trimer immunogen and uses thereof. Background Art

[0002] Severe acute respiratory syndrome coronavirus (SARS-CoV) and the novel coronavirus (SARS-CoV-2) both belong to the genus Sabaecoronavirus. The key mechanism by which these two viruses infect their hosts is the interaction between the receptor binding domain (RBD) on the viral surface spike protein (S protein) and the human angiotensin-converting enzyme 2 (ACE2), mediating viral entry into host cells. Research suggests that Sabaecoronavirus's ability to bind to ACE2 is a relatively ancient property, likely prevalent in a common ancestor in Asia and neighboring regions.

[0003] Bats are natural reservoirs of coronaviruses, and numerous Sabae coronaviruses have been isolated from them. The diversity of viruses carried by bats and their potential for cross-species transmission have become a hot topic of research. Previous studies have found that RaTG13, isolated from the Chinese horseshoe bat (Rhinolophus sinicus), is highly similar to the full genome sequence of SARS-CoV-2, with a sequence identity of 96.2%. As more bat coronaviruses have been discovered, a study by Sarah Temmam et al. revealed that BANAL-52 and BANAL-236, with genome sequences highly similar to SARS-CoV-2, were detected in 645 bats captured in caves in northern Laos. The RBD region of BANAL-52 shares 97.4% sequence identity with SARS-CoV-2, while the RBD of BANAL-236 shares 96.9% sequence identity. Further functional experiments have demonstrated that these viruses efficiently bind to hACE2 and mediate host cell infection.

[0004] Furthermore, Jing Wang et al. analyzed rectal samples from 149 bats collected between 2015 and 2019 and found that the bat-derived coronavirus BtSY1 shared 93% full-genome sequence similarity with SARS-CoV, with a high 98.13% sequence identity in the N-terminal domain (NTD), but only 88.61% similarity in the RBD. They also discovered a bat-derived coronavirus, BtSY2, that shared 92% full-genome sequence identity with SARS-CoV-2, 95.15% sequence identity in the NTD, and 93.70% similarity in the RBD. Notably, BtSY2 differed in the RBD at only five key amino acid positions: T372A, L486F, H489Q, Y501N, and N519H. These molecular features suggest that BtSY2 shares a high degree of similarity with SARS-CoV-2. Functional analysis further confirmed that BtSY2 can utilize the hACE2 receptor to mediate cell invasion, and its binding stability is highly similar to that of the SARS-CoV-2 prototype strain.

[0005] For most SARS-CoV-2 RBD regions, a single amino acid mutation can enhance their binding ability to existing ACE2 homologs and even confer the potential for cross-species transmission. For example, the N501Y mutation in SARS-CoV-2 enhances its affinity for hACE2, while significantly reducing its binding ability in SARS-CoV.

[0006] In November 2021, the Omicron variant was first discovered in South Africa. There are a large number of mutations on its S protein, which has aroused widespread concern in the scientific community about its ability to escape the immune system. Research by Qian Wang and others has shown that compared with the D614G mutant, the immune escape ability of antibodies against variants such as BQ.1, BQ.1.1, XBB and XBB.1 is significantly enhanced. In the sera of vaccine recipients and previously infected people, the decrease in neutralization ability can reach 13-81 times in the BQ subvariant and 66-155 times in the XBB subvariant. This phenomenon indicates that the BQ and XBB subvariants may pose a serious challenge to the immune protection ability of existing new coronavirus vaccines.

[0007] At the molecular level, the key amino acid mutation sites of the XBB subvariant include Q183E, V445P, and F486S, while the key mutation sites of BQ.1.1 include R346T, K444T, and N460K. Among them, the R346T mutation is an important adaptive mutation that appeared during the evolution of the BA.4 / BA.5 variants. The XBB and BQ.1.1 variants mainly change the three-dimensional structure and charge distribution of the S protein through key amino acid mutations in the RBD and NTD regions, thereby reducing the binding ability of neutralizing antibodies and leading to immune escape.

[0008] SARS-CoV-2 Omicron variants continue to evolve. As of now, SARS-CoV-2 serotypes can be divided into the following six categories:

[0009] Serotype I: covers all variants before Omicron (such as Alpha, Beta, Gamma, Delta, etc.);

[0010] Serotype II: includes Omicron BA.1 and BA.1.1 subvariants;

[0011] Serotype III: includes Omicron BA.2, BA.2.12.1, and BA.2.75 subvariants;

[0012] Serotype IV: covers Omicron BA.5, BF.7, BQ.1 and BQ.1.1 subvariants;

[0013] Serotype V: includes Omicron XBB and XBB.1.5 subvariants;

[0014] Serotype VI: includes the Omicron BA.2.86 subvariant.

[0015] In order to effectively respond to the continued evolution of SARS-CoV-2Omicron variants and the possible cross-species transmission risk of other Sabeicoronavirus viruses in the future, it is necessary to develop a vaccine that can provide broad-spectrum and effective immune protection against Sabeicoronavirus viruses to reduce the risk of potential Sabeicoronavirus pandemics in the future. Summary of the Invention

[0016] Purpose of the Invention

[0017] The object of the present invention is to provide a recombinant trimeric immunogen having a broad-spectrum and high-level immune protection effect against Sabei coronavirus viruses, a related vaccine product and its use.

[0018] Solution

[0019] To achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0020] In a first aspect, the present invention provides a recombinant trimeric immunogen of Sabei coronavirus, wherein the recombinant trimeric immunogen comprises an amino acid sequence arranged in the pattern shown in formula (I):

[0021] (AB)-C1-(AB)1-C2-(AB)2

[0022] (I)

[0023] In formula (I):

[0024] (AB) and (AB)1 each independently represent the S protein RBD domain of the novel coronavirus prototype strain or variant strain or its functionally active fragment, or their derivative sequences;

[0025] (AB)2 represents the S protein RBD domain of severe acute respiratory syndrome coronavirus or its functionally active fragment, or a derivative sequence thereof;

[0026] 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.

[0027] In a possible embodiment I, in formula (I):

[0028] (AB) represents the RBD domain of the S protein of the novel coronavirus prototype strain or a functionally active fragment thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity thereto; preferably, (AB) represents the amino acid sequence as shown in SEQ ID NO: 1;

[0029] (AB)1 represents the RBD domain of the S protein of the novel coronavirus Omicron variant XBB subtype or a functionally active fragment 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; preferably, (AB)1 represents the amino acid sequence as shown in SEQ ID NO: 2;

[0030] (AB)2 represents the S protein RBD domain of severe acute respiratory syndrome coronavirus or a functionally active fragment 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; preferably, (AB)2 represents the amino acid sequence as shown in SEQ ID NO:3;

[0031] Preferably, n=0, 1, 2 or 3;

[0032] Preferably, the recombinant trimeric immunogen comprises the amino acid sequence shown in SEQ ID NO:9.

[0033] In a possible embodiment II, in formula (I):

[0034] (AB) represents the RBD domain of the S protein of the novel coronavirus Omicron variant XBB subtype or a functionally active fragment 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; preferably, (AB) represents the amino acid sequence as shown in SEQ ID NO: 4;

[0035] (AB)1 represents the S protein RBD domain of the novel coronavirus Omicron variant BQ.1.1 subtype or a functionally active fragment 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; preferably, (AB)1 represents the amino acid sequence as shown in SEQ ID NO:5;

[0036] (AB)2 represents the S protein RBD domain of severe acute respiratory syndrome coronavirus or a functionally active fragment 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; preferably, (AB)2 represents the amino acid sequence as shown in SEQ ID NO:3;

[0037] Preferably, n=0, 1, 2 or 3;

[0038] Preferably, the recombinant trimeric immunogen comprises the amino acid sequence shown in SEQ ID NO:10.

[0039] In a possible embodiment III, in formula (I):

[0040] (AB) represents the RBD domain of the S protein of the novel coronavirus Omicron variant XBB subtype or a functionally active fragment 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; preferably, (AB) represents the amino acid sequence as shown in SEQ ID NO: 4;

[0041] (AB)1 represents the RBD domain of the S protein of the novel coronavirus Omicron variant JN.1 subtype or a functionally active fragment 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; preferably, (AB)1 represents the amino acid sequence as shown in SEQ ID NO:6;

[0042] (AB)2 represents the S protein RBD domain of severe acute respiratory syndrome coronavirus or a functionally active fragment 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; preferably, (AB)2 represents the amino acid sequence as shown in SEQ ID NO:3;

[0043] Preferably, n=0, 1, 2 or 3;

[0044] Preferably, the recombinant trimeric immunogen comprises the amino acid sequence shown in SEQ ID NO:11.

[0045] In a possible embodiment IV, in formula (I):

[0046] (AB) represents the S protein RBD domain of the novel coronavirus Omicron variant BQ.1.1 subtype or a functionally active fragment 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; preferably, (AB) represents the amino acid sequence as shown in SEQ ID NO: 7;

[0047] (AB)1 represents the RBD domain of the S protein of the novel coronavirus Omicron variant JN.1 subtype or a functionally active fragment 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; preferably, (AB)1 represents the amino acid sequence as shown in SEQ ID NO:6;

[0048] (AB)2 represents the S protein RBD domain of severe acute respiratory syndrome coronavirus or a functionally active fragment 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; preferably, (AB)2 represents the amino acid sequence as shown in SEQ ID NO:3;

[0049] Preferably, n=0, 1, 2 or 3;

[0050] Preferably, the recombinant trimeric immunogen comprises the amino acid sequence shown in SEQ ID NO:12.

[0051] Preferably, the N-terminus of the recombinant trimeric immunogen further includes a signal peptide, and the signal peptide preferably has an amino acid sequence as shown in SEQ ID NO: 13; and / or, the C-terminus further includes a tag sequence, and the tag is preferably a His tag.

[0052] In a second aspect, the present invention provides a polynucleotide encoding the recombinant trimeric immunogen as described in the first aspect above;

[0053] Optionally, the polynucleotide is a DNA molecule, preferably, the DNA molecule comprises or consists of a DNA sequence as shown in one of SEQ ID NOs: 14-17;

[0054] Optionally, the polynucleotide is an mRNA molecule; preferably, the mRNA molecule comprises or consists of an RNA sequence corresponding to a DNA sequence as shown in one of SEQ ID NOs: 14-17.

[0055] In a third aspect, the present invention provides a nucleic acid construct, an expression vector or a host cell, comprising the polynucleotide described in the second aspect above.

[0056] In a fourth aspect, the present invention provides the use of the recombinant trimeric immunogen as described in the first aspect, the polynucleotide as described in the second aspect, or the nucleic acid construct, expression vector or host cell as described in the third aspect in the preparation of a medicament for preventing and / or treating Sabei coronavirus infection;

[0057] Optionally, the drug is a vaccine;

[0058] Optionally, the Sabae coronavirus is selected from the group consisting of: novel coronavirus, severe respiratory syndrome coronavirus and other SARS-related coronaviruses;

[0059] Further optionally, the novel coronavirus is selected from: SARS-CoV-2 prototype strain, D614G mutant strain and Omicron lineages BA.2, BQ.1.1, XBB, JN.1, KP.2, KP.3.1.1;

[0060] Further optionally, the other SARS-related coronaviruses are selected from: WIV1, BtSY2 and Pangolin GD.

[0061] In a fifth aspect, the present invention provides an immunogenic composition comprising the recombinant trimeric immunogen as described in the first aspect above, the polynucleotide as described in the second aspect above, or the nucleic acid construct, expression vector or host cell as described in the third aspect above, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

[0062] In some preferred embodiments, the immunogenic composition is a Sabei coronavirus recombinant protein vaccine, which comprises the recombinant trimeric immunogen and an adjuvant as described in the first aspect above;

[0063] Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant.

[0064] In other preferred embodiments, the immunogenic composition is a Sabei coronavirus DNA vaccine, comprising:

[0065] (1) a eukaryotic expression vector; and

[0066] (2) a DNA sequence encoding the recombinant trimeric immunogen as described in the first aspect above, constructed and incorporated into the eukaryotic expression vector;

[0067] Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

[0068] In other preferred embodiments, the immunogenic composition is a Sabei coronavirus mRNA vaccine, and the mRNA vaccine comprises:

[0069] (I) an mRNA sequence encoding the recombinant trimeric immunogen as described in the first aspect above; and

[0070] (II) Lipid nanoparticles.

[0071] In other preferred embodiments, the immunogenic composition is a Sabei coronavirus-viral vector vaccine comprising:

[0072] (1) viral backbone vectors; and

[0073] (2) a DNA sequence encoding the recombinant trimeric immunogen as described in the first aspect above, constructed into the viral backbone vector;

[0074] Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.

[0075] In other preferred embodiments, the immunogenic composition is a Sabei coronavirus nanoparticle vaccine, wherein the recombinant trimeric immunogen as described in the first aspect above is covalently linked to the self-assembling nanoparticle protein and self-assembled into nanoparticles, so that the recombinant trimeric immunogen is presented on the surface of the nanoparticles;

[0076] Optionally, the carrier of the nanoparticle is ferritin; further optionally, the recombinant trimer immunogen is connected to ferritin via a linker, or is covalently linked via a SpyTag / SpyCatcher connection system.

[0077] For the above immunogenic composition, preferably, the immunogenic composition is in the form of a nasal spray, an oral preparation, a suppository or a parenteral preparation;

[0078] Further preferably, the nasal spray is selected from aerosols, sprays and powder sprays;

[0079] Further preferably, the oral preparation is selected from tablets (e.g., sublingual tablets), powders (e.g., powders), pills (e.g., pellets), granules (e.g., fine granules), soft / hard capsules, film-coated preparations, and ointments;

[0080] More preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, a liquid for external use, or an injectable preparation (eg, a push-in preparation).

[0081] In a sixth aspect, the present invention provides a kit comprising the recombinant trimeric immunogen described in the first aspect above, the polynucleotide described in the second aspect above, the nucleic acid construct, expression vector or host cell described in the third aspect above and / or the immunogenic composition described in the fifth aspect above.

[0082] In a preferred embodiment, the kit comprises:

[0083] (1) the recombinant trimeric immunogen, the polynucleotide encoding the same, and / or the nucleic acid construct, expression vector or host cell comprising the polynucleotide as described in the feasible embodiment 1 of the first aspect above; and

[0084] (2) The recombinant trimeric immunogen, the polynucleotide encoding the same and / or the nucleic acid construct, expression vector or host cell comprising the polynucleotide as described in the feasible embodiment IV of the first aspect above.

[0085] In a seventh aspect, the present application provides a method for preventing and / or treating a Sabei coronavirus infectious disease, the method comprising administering to a subject in need thereof a preventive and / or therapeutically effective amount of the following substances:

[0086] The recombinant trimeric immunogen as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct, expression vector or host cell as described in the third aspect above and / or the immunogenic composition as described in the fifth aspect above.

[0087] Optionally, the Sabae coronavirus is selected from the group consisting of: novel coronavirus, severe respiratory syndrome coronavirus and other SARS-related coronaviruses;

[0088] Further optionally, the novel coronavirus is selected from: SARS-CoV-2 prototype strain, D164G mutant strain and Omicron lineage variant strains BA.2, BQ.1.1, XBB, JN.1, KP.2, KP.3.1.1;

[0089] Further optionally, the other SARS-related coronaviruses are selected from: WIV1, BtSY2 and Pangolin GD.

[0090] 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.

[0091] Beneficial effects

[0092] The recombinant trimeric immunogen of the Sabei coronavirus provided by the present invention can not only provide balanced and efficient immune protection for the SARS-CoV-2 prototype strain, the D164G mutant strain, and the latest popular Omicron lineage variant strains, but also can efficiently stimulate the body to produce high-level neutralizing antibodies against non-SARS-CoV-2 Sabei coronaviruses (especially SARS, WIV1, BtSY2, and Pangolin GD), thereby providing a broad-spectrum and efficient immune protection effect for Sabei coronavirus viruses, achieving unexpected technical effects. Based on the above-mentioned excellent immune performance, the recombinant trimeric immunogen of the present invention has great potential to become the core immunogen of a broad-spectrum vaccine for Sabei coronavirus viruses, providing more comprehensive and effective immune protection against such viral infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] 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.

[0094] Figure 1Schematic diagram of the structure of the trimeric immunogen PT-XBB-SARS and XBB-PT-SARS constructs constructed in Example 1.

[0095] Figure 2 This is a graph showing the molecular sieve chromatography curves of the trimeric immunogens PT-XBB-SARS and XBB-PT-SARS expressed by 293F cells and the SDS-PAGE identification results of the eluate at the elution peak, as described in Example 1.

[0096] Figure 3 This is the structural diagram of the trimeric immunogen PT-XBB-SARS predicted by the AlphaFold method.

[0097] Figure 4 The results of neutralizing antibody titer detection against pseudoviruses of SARS-CoV-2Omicron subtype variants induced by the trimeric immunogen PT-XBB-SARS are shown; the horizontal axis shows the group, and the vertical axis shows the neutralizing antibody titer (pVNT 50 ).

[0098] Figure 5 The results of neutralizing antibody titers against pseudoviruses of SARS-CoV, BtSY2, Pangolin GD, and WIV1 induced by the trimeric immunogen PT-XBB-SARS are shown; the horizontal axis shows the group, and the vertical axis shows the neutralizing antibody titer (pVNT 50 ).

[0099] Figure 6 It is a schematic diagram of the structures of the trimeric immunogens XBB-BQ.1.1-SARS, XBB-JN.1-SARS and BQ.1.1-JN.1-SARS constructs constructed in Example 4.

[0100] Figure 7 This is a diagram showing the molecular sieve chromatography curves of the trimeric immunogens XBB-BQ.1.1-SARS, XBB-JN.1-SARS and BQ.1.1-JN.1-SARS expressed by 293F cells and the SDS-PAGE identification results of the eluate at their elution peaks, as described in Example 4.

[0101] Figure 8The results of neutralizing antibody titer detection against pseudoviruses of various subtype variants of SARS-CoV-2Omicron induced by the trimeric immunogens XBB-BQ.1.1-SARS, XBB-JN.1-SARS and BQ.1.1-JN.1-SARS and the mixed immunogen composed of PT-XBB-SARS and BQ.1.1-JN.1-SARS (i.e., PXS&BJS group); the horizontal axis shows the group, and the vertical axis shows the neutralizing antibody titer (pVNT 50 ).

[0102] Figure 9 The results of neutralizing antibody titers against pseudoviruses of SARS-CoV, BtSY2, Pangolin GD and WIV1 induced by the trimeric immunogens XBB-BQ.1.1-SARS, XBB-JN.1-SARS and BQ.1.1-JN.1-SARS, as well as the mixed immunogen composed of PT-XBB-SARS and BQ.1.1-JN.1-SARS (i.e., PXS&BJS group); the horizontal axis shows the group, and the vertical axis shows the neutralizing antibody titer (pVNT 50 ).

[0103] Figure 10 This is a radar chart of the neutralizing activity of each immunogen against SARS-CoV-2Omicron subtype variants, SARS-CoV, and SARS-related coronaviruses BtSY2, Pangolin GD, and WIV1. DETAILED DESCRIPTION

[0104] 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.

[0105] 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.

[0106] 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.

[0107] Example 1: Design and protein expression of RBD trimer immunogens PT-XBB-SARS and XBB-PT-SARS

[0108] In this example, the Sabella coronavirus RBD trimer immunogens PT-XBB-SARS and XBB-PT-SARS were designed, their expression plasmids were constructed, and protein expression and purification were performed. The specific scheme is as follows.

[0109] Design of trimer immunogen and construction of its expression plasmid:

[0110] (1) The RBD domain sequence of the SARS-CoV-2 prototype strain as shown in SEQ ID NO: 1, the RBD domain sequence of the SARS-CoV-2 Omicron XBB variant strain as shown in SEQ ID NO: 2, and the RBD domain sequence of SARS-CoV as shown in SEQ ID NO: 3 were directly connected in series (obtaining the amino acid sequence as shown in SEQ ID NO: 9), and the kozak sequence (GCCACC) and the signal peptide (as shown in SEQ ID NO: 13) were added to the N-terminus, and the 6×His tag and the stop codon were added to the C-terminus to obtain the immunogen PT-XBB-SARS construct (its structural schematic is shown in Figure 1 ); codon optimization was performed on the coding nucleic acid sequence of the PT-XBB-SARS construct according to the codon preference of the 293F cell expression system, and the optimized coding nucleic acid was cloned into the pCAGGS vector to obtain the expression plasmid pCAGGS-PT-XBB-SARS of the immunogen PT-XBB-SARS;

[0111] (2) The RBD domain sequence of the SARS-CoV-2Omicron XBB variant, the RBD domain sequence of the SARS-CoV-2 prototype strain, and the RBD domain sequence of SARS-CoV were directly concatenated (to obtain the amino acid sequence shown in SEQ ID NO: 8), and the kozak sequence (GCCACC) and the signal peptide (shown in SEQ ID NO: 13) were added to the N-terminus, and the 6×His tag and the stop codon were added to the C-terminus to obtain the immunogen XBB-PT-SARS construct (its structural schematic is shown in Figure 1 ); the coding nucleic acid sequence of the XBB-PT-SARS construct was codon-optimized according to the codon preference of the 293F cell expression system, and the optimized coding nucleic acid was cloned into the pCAGGS vector to obtain the expression plasmid pCAGGS-XBB-PT-SARS of the immunogen XBB-PT-SARS.

[0112] Protein expression and purification:

[0113] (1) The expression plasmids of the trimeric immunogens PT-XBB-SARS and XBB-PT-SARS constructed above were transfected into 293F cells respectively. After 5 days, the supernatant was collected, centrifuged to remove the precipitate, and then filtered through a 0.22 μm filter membrane to further remove impurities.

[0114] (2) purifying the obtained cell supernatant by nickel affinity column chromatography;

[0115] The conditions and procedures for nickel affinity column chromatography are as follows: a HisTrap excel (cytiva) chromatography column with a column volume of 5 ml CV and a flow rate of 1-2 ml / min; pretreatment: first rinse with double distilled water for 5-10 CVs; equilibration: rinse with Binding buffer (1×PBS buffer, pH 7.4) for 5-10 CVs; loading: take 293F suspension cell culture filtered through a 0.22 μm filter membrane and load it onto the equilibrated chromatography column; washing: after loading, first rinse with Binding buffer for at least 4 CVs until the UV baseline is flat, then elute with elution buffer of different concentrations (1×PBS + 1M imidazole, pH 7.4) until the UV baseline is flat, collect the eluted fractions in separate tubes, and perform nickel affinity column chromatography on them separately. According to the analysis results of the SDS-PAGE gel electrophoresis diagram, the target protein in the collection tubes is combined and concentrated for the next step of gel filtration chromatography.

[0116] 3) further purifying the concentrated protein by gel filtration chromatography (i.e., molecular sieve chromatography);

[0117] The conditions and procedures for gel filtration chromatography are as follows:

[0118] Using Superdex TM 200Increase 10 / 300GL (cytiva) gel filtration chromatography column, column volume CV is 24 ml, flow rate is 0.4 ml / min; pretreatment: rinse with double distilled water for 1 CV; equilibration: equilibrate with Binding buffer (1×PBS buffer, pH 7.4) for 1 CV; loading: load the concentrated protein onto the gel column through a loop; collection: collect the target protein, and the target protein can be combined and concentrated to obtain the trimeric immunogen protein PT-XBB-SARS and XBB-PT-SARS.

[0119] The molecular sieve chromatography curves of the trimeric immunogen proteins PT-XBB-SARS and XBB-PT-SARS are as follows Figure 2 As shown, Figure 2The results showed that both PT-XBB-SARS and XBB-PT-SARS had a main elution peak at an elution volume of about 12.4 mL. The eluates at the main elution peak were collected and analyzed by SDS-PAGE under reducing (+DTT) or non-reducing conditions (-DTT). The results are shown in Figure 2 . Figure 2 The results showed that the eluted proteins at the main elution peak were all around 76 KDa in size, consistent with their corresponding theoretical molecular weight. Moreover, after treatment with the reducing agent DTT, the molecular weight of the protein did not change, indicating that the trimeric immunogens PT-XBB-SARS and XBB-PT-SARS did not contain disulfide bonds and had good stability. In addition, the SDS-PAGE electrophoresis band was single, indicating that the purified protein had a high purity.

[0120] In addition, according to the absorption peak at 280nm during the protein purification process, the protein expression level of PT-XBB-SARS was significantly higher than that of XBB-PT-SARS, which suggests that placing the SARS-CoV-2 prototype strain first in the design of trimer immunogens may help increase the expression of antigen proteins.

[0121] According to the above method, four purified dimeric immunogen proteins BA.2-SARS, PT-SARS, PT-PT, and SARS-SARS (which correspond to BA.2-S, CS, CC, and SS in CN 202311065664.1, respectively) were obtained, which will be used as control immunogens below.

[0122] Example 2: Structural prediction of the trimeric immunogen PT-XBB-SARS

[0123] After removing the signal peptide and his tag from the amino acid sequence of the trimeric immunogen PT-XBB-SARS, the AlphaFold deep learning model, which has been trained on a large number of protein structure datasets, was used to predict the three-dimensional structure of the target protein. The predicted distances between residues were used to generate a distance matrix to predict the three-dimensional coordinates of the protein atoms. The structure of the trimeric immunogen PT-XBB-SARS expressed in Example 1 was predicted using the AlphaFold method (https: / / alphafold.ebi.ac.uk / ). The results are shown in Figure 2. Figure 3 .

[0124] Figure 3 It shows that in the trimeric immunogen PT-XBB-SARS, each RBD unit is stacked through the core motif, while fully exposing the external motif and receptor binding motif (RBM); this structural feature ensures the full presentation of the antigenic epitope, thereby enhancing its immunogenicity.

[0125] Example 3: Immunogenicity Detection of the Trimeric Immunogen PT-XBB-SARS

[0126] In order to detect the immunogenicity of the trimeric immunogen PT-XBB-SARS, we used the purified immunogen protein PT-XBB-SARS obtained in Example 1 as an immunogen to immunize BALB / c mice and evaluated the immunogenicity of PT-XBB-SARS by pseudovirus neutralization experiments.

[0127] Vaccine preparation

[0128] The trimeric immunogen protein PT-XBB-SARS and the dimeric immunogen proteins BA.2-SARS, PT-SARS, PT-PT, and SARS-SARS used as controls were diluted to 20 μg / ml with PBS, respectively. The diluted immunogens were then fully mixed and thoroughly emulsified with an MF59-like adjuvant, AddaVaxTM adjuvant, at a volume ratio of 1:1 to prepare the vaccine. At the same time, the PBS solution was mixed with AddaVaxTM adjuvant as a negative control group.

[0129] Immunization regimen

[0130] BALB / c mice (female, 6-8 weeks old, purchased from Weitonglihua Company) were immunized by intramuscular injection with the vaccine obtained according to the above method. The experiment was divided into 6 groups, with 10 mice in each group. For all mice, the first immunization was performed on day 0, and blood was collected after the first immunization on day 19; the second immunization was performed on day 21, and blood was collected after the second immunization on day 35; the third immunization was performed on day 42, and blood was collected after the third immunization on day 56; the inoculation volume for each mouse for each immunization was 100 μL (containing 2 μg of immunogen protein).

[0131] The experimental groups and immunization schemes are shown in Table 1.

[0132] Table 1. Grouping and immunization scheme of mice immunized with each immunogen

[0133]

[0134] The blood collected after each immunization was centrifuged at 12,000 rpm for 10 min to collect the serum, which was inactivated at 56°C for 30 min to inactivate complement and then stored at -80°C for subsequent experiments.

[0135] Pseudovirus neutralization experiment

[0136] In this example, the neutralizing antibody titers (pVNT) of the mouse serum after the third immunization (i.e., collected on the 56th day) against the novel coronavirus Omicron variant D614G, BA.2, BQ.1.1, XBB, JN.1, KP.2, KP.3.1.1 subtypes, SARS-CoV, and pseudoviruses of SARS-related coronaviruses BtSY2, Pangolin GD, and WIV1 were detected. 50 ).

[0137] The packaging of the pseudoviruses of each subtype variant of the novel coronavirus Omicron, SARS-CoV, BtSY2, PangolinGD, and WIV used in this example was all carried out in a Class II biosafety laboratory by carrying the spike proteins of the corresponding viruses on the vesicular stomatitis virus (VSV) backbone. For the preparation method, see Zhao X, Zheng A, Li D, Zhang R, Sun H, Wang Q, Gao GF, Han P, Dai L. Neutralisation of ZF2001-elicited antiserato SARS-CoV-2 variants. Lancet Microbe. 2021 Oct; 2(10): e494. doi: 10.1016 / S2666-5247(21)00217-2. Epub 2021 Aug 20. PMID: 34458880; PMCID: PMC8378832.

[0138] The method for detecting the titer of pseudovirus neutralizing antibodies against SARS-CoV and SARS-CoV-2Omicron subtype variants is as follows:

[0139] In a 96-well plate, immune mouse serum was diluted in DMEM medium (containing 10% fetal bovine serum) in a 2-fold serial dilution, with an initial dilution of 1:40, and a total of 10 serial dilutions were set up. Similarly, pseudovirus was diluted to 2000 TU / 100 μl in DMEM medium (containing 10% fetal bovine serum). Subsequently, the diluted immune mouse serum and diluted pseudovirus were mixed at a volume ratio of 1:1. Blank medium mixed with pseudovirus served as a negative control (NC), and blank medium without pseudovirus served as a blank control (MOCK). The plates were incubated at 37°C for 1 hour. The immune mouse serum-pseudovirus mixture was then transferred to a 96-well plate filled with Vero E6 cells and incubated at 37°C for 15 hours. The number of positive cells was detected and counted using a CQ1 confocal cell imager (Yokogawa). Then, a fitting curve was drawn in GraphPad Prism software, and the reciprocal of the serum dilution corresponding to 50% neutralization was calculated, which was the neutralization titer (pVNT). 50 .

[0140] The method for detecting the titer of neutralizing antibodies against pseudoviruses of SARS-related coronaviruses BtSY2, Pangolin GD, and WIV is as follows:

[0141] In a 96-well plate, immune mouse serum was diluted in DMEM medium (containing 10% fetal bovine serum) in a 2-fold serial dilution, with an initial dilution of 1:40, and a total of 10 gradients were set. Similarly, pseudovirus was diluted to 1000TU / 100μl in DMEM medium (containing 10% fetal bovine serum). Subsequently, the diluted immune mouse serum and diluted pseudovirus were mixed in a volume ratio of 1:1, and blank medium mixed with pseudovirus was used as a negative control (NC), and blank medium without pseudovirus was used as a blank control (MOCK). The plates were incubated at 37°C for 1 hour. Then, the immune mouse serum-pseudovirus mixture was transferred to a 96-well plate filled with Vero E6-ACE2 cells. After incubation at 37°C for 15 hours, the positive cells were detected and counted using a CQ1 confocal cell imager (Yokogawa). Then, a fitting curve was drawn in GraphPad Prism software, and the reciprocal of the serum dilution corresponding to 50% neutralization was calculated, which was the neutralization titer (pVNT). 50 .

[0142] The neutralizing antibody titer test results of each immune mouse serum against pseudoviruses of each subtype variant of SARS-CoV-2Omicron are as follows Figure 4 shown.

[0143] Figure 4 The results show:

[0144] 1) The trimeric immunogen PT-XBB-SARS can induce mice to produce high-titer neutralizing antibodies against the SARS-CoV-2 D614G mutant and various Omicron subtype variants (including BA.2, BQ.1.1, XBB, JN.1, KP.2 and KP.3.1.1), with the neutralizing antibody titer exceeding 10 3 ;The results show that the trimeric immunogen PT-XBB-SARS can exhibit a high immune protection effect against all subtype variants of SARS-CoV-2Omicron.

[0145] 2) At the same immune dose, the neutralizing antibody levels induced by the trimeric immunogen PT-XBB-SARS against the Omicron variants BQ.1.1, XBB, JN.1, KP.2 and KP.3.1.1 were significantly enhanced compared to the dimeric immunogens BA.2-SARS, PT-SARS, PT-PT, and SARS-SARS. In addition, although the neutralizing antibody levels induced by PT-XBB-SARS against the SARS-CoV-2 variants D614G and BA.2 were comparable to or slightly lower than those of some dimeric antigens, it still induced a relatively high level of neutralizing antibodies, which was fully capable of meeting the immune protection requirements against these two virus strains. Overall, the neutralizing activity level of PT-XBB-SARS against the SARS-CoV-2Omicron subtype variants was still significantly better than that of the dimeric antigens of the prior art, which represents a significant advancement of the trimeric immunogen PT-XBB-SARS of the present invention over the prior art.

[0146] The neutralizing antibody titer test results of each immune mouse serum against SARS-CoV and BtSY2, Pangolin GD, WIV pseudovirus are as follows Figure 5 shown.

[0147] Figure 5 The results show:

[0148] I) The trimeric immunogen PT-XBB-SARS was able to induce mice to produce high titer neutralizing antibodies against the above viruses, with the neutralizing antibody titers exceeding 10 3 , some even more than 10 4 (WIV1, SARS-CoV), 10 5 (PangolinGD); The results showed that the trimeric immunogen PT-XBB-SARS also showed a high immune protection effect against non-SARS-CoV-2 Sabei coronavirus;

[0149] II) At the same immunization dose, the trimeric immunogen PT-XBB-SARS induced neutralizing antibody levels against the aforementioned non-SARS-CoV-2 sabemicoronaviruses, either comparable to or significantly enhanced compared to the dimeric immunogen. Overall, the neutralizing activity of PT-XBB-SARS against non-SARS-CoV-2 sabemicoronaviruses was still significantly superior to that of the dimeric antigens of the prior art, representing a significant advantage of the trimeric immunogen PT-XBB-SARS of the present invention over the prior art.

[0150] From the above results, it can be seen that the trimeric immunogen PT-XBB-SARS showed significantly better neutralizing activity than the existing dimeric immunogens in dealing with various subtype variants of SARS-CoV-2Omicron and non-SARS-CoV-2 Sabei coronaviruses, and demonstrated a broad-spectrum and highly effective immune protection effect across the entire Sabei coronavirus genus.

[0151] Example 4: Design and protein expression of RBD trimer immunogens XBB-BQ.1.1-SARS, XBB-JN.1-SARS, and BQ.1.1-JN.1-SARS

[0152] In this example, Sabella coronavirus RBD trimer immunogens XBB-BQ.1.1-SARS, XBB-JN.1-SARS and BQ.1.1-JN.1-SARS were designed respectively, and their expression plasmids were constructed and protein expression and purification were performed with reference to the method described in Example 1.

[0153] The schematic diagram of the structure of the constructs of the trimeric immunogens XBB-BQ.1.1-SARS, XBB-JN.1-SARS and BQ.1.1-JN.1-SARS is shown in Figure 6 As shown, the sequence is as follows:

[0154] XBB-BQ.1.1-SARS: The RBD domain sequence of the SARS-CoV-2 Omicron XBB variant as shown in SEQ ID NO:4, the RBD domain sequence of the SARS-CoV-2 Omicron BQ.1.1 variant as shown in SEQ ID NO:5, and the RBD domain sequence of SARS-CoV as shown in SEQ ID NO:3 are directly concatenated to obtain the amino acid sequence as shown in SEQ ID NO:10, which is the amino acid sequence of the immunogen XBB-BQ.1.1-SARS; the Kozak sequence (GCCACC) and the signal peptide (as shown in SEQ ID NO:13) are added to the N-terminus, and the 6×His tag and stop codon are added to the C-terminus to obtain the construct of the immunogen XBB-BQ.1.1-SARS;

[0155] XBB-JN.1-SARS: The RBD domain sequence of the SARS-CoV-2 Omicron XBB variant as shown in SEQ ID NO:4, the RBD domain sequence of the SARS-CoV-2 Omicron JN.1 variant as shown in SEQ ID NO:6, and the RBD domain sequence of SARS-CoV as shown in SEQ ID NO:3 were directly concatenated to obtain the amino acid sequence as shown in SEQ ID NO:11, which is the amino acid sequence of the immunogen XBB-JN.1-SARS; the Kozak sequence (GCCACC) and the signal peptide (as shown in SEQ ID NO:13) were added to the N-terminus, and a 6×His tag and a stop codon were added to the C-terminus to obtain the construct of the immunogen XBB-JN.1-SARS;

[0156] BQ.1.1-JN.1-SARS: The RBD domain sequence of the SARS-CoV-2Omicron BQ.1.1 variant as shown in SEQ ID NO:7, the RBD domain sequence of the SARS-CoV-2Omicron JN.1 variant as shown in SEQ ID NO:6, and the RBD domain sequence of SARS-CoV as shown in SEQ ID NO:3 are directly concatenated to obtain the amino acid sequence shown in SEQ ID NO:12, which is the amino acid sequence of the immunogen BQ.1.1-JN.1-SARS; the kozak sequence (GCCACC) and the signal peptide (as shown in SEQ ID NO:13) are added to its N-terminus, and a 6×His tag and a stop codon are added to its C-terminus to obtain the construct of the immunogen BQ.1.1-JN.1-SARS.

[0157] The molecular sieve chromatography curves of the trimeric immunogen proteins XBB-BQ.1.1-SARS, XBB-JN.1-SARS and BQ.1.1-JN.1-SARS expressed according to the method described in Example 1 are as follows: Figure 7 shown. Figure 7 The results showed that XBB-BQ.1.1-SARS, XBB-JN.1-SARS and BQ.1.1-JN.1-SARS all had a main elution peak at an elution volume of about 12.4 mL. The eluates at the main elution peak were collected and analyzed by SDS-PAGE under reducing (+DTT) or non-reducing conditions (-DTT). The results are shown in Figure 7 , Figure 7The results showed that the eluted proteins at the main elution peak were all around 76 KDa in size, consistent with their corresponding theoretical molecular weight. Furthermore, after treatment with the reducing agent DTT, the protein molecular weight did not change, indicating that the three trimeric immunogens did not contain disulfide bonds and had good stability. Furthermore, the SDS-PAGE electrophoresis bands were single, indicating that the purified proteins had a high purity.

[0158] From the above results, it can be seen that the three trimeric immunogenic proteins were successfully expressed in this example, and it was observed that they all showed high yield and easy expression characteristics, similar to PT-XBB-SARS.

[0159] Example 5: Immunogenicity testing of trimeric immunogens XBB-BQ.1.1-SARS, XBB-JN.1-SARS, BQ.1.1-JN.1-SARS, and a mixed immunogen composed of PT-XBB-SARS and BQ.1.1-JN.1-SARS

[0160] To test the immunogenicity of the trimeric immunogens XBB-BQ.1.1-SARS, XBB-JN.1-SARS, and BQ.1.1-JN.1-SARS, we immunized BALB / c mice with the purified proteins obtained in Example 4 and evaluated their immunogenicity using pseudovirus neutralization assays. The vaccine preparation and mouse immunization protocols, as well as the pseudovirus neutralization assay protocols, were similar to those described in Example 3.

[0161] At the same time, we also explored a mixed immunization strategy; specifically, PT-XBB-SARS and BQ.1.1-JN.1-SARS were mixed in a mass ratio of 1:1 as a mixed immunogen (also known as PXS&BJS immunization group). The immunization scheme of this mixed immunogen was basically the same as that of the single immunogen, except that the single immunization dose was different. The single immunization dose of this mixed immunogen was divided into two types: 1μg group (i.e., 1μg PT-XBB-SARS was mixed with 1μg BQ.1.1-JN.1-SARS and diluted to 100μl, and each mouse was inoculated with 100μl of the above-mentioned diluted mixed immunogen solution each time) and 2μg group (i.e., 2μg PT-XBB-SARS was mixed with 2μg BQ.1.1-JN.1-SARS and diluted to 100μl, and each mouse was inoculated with 100μl of the above-mentioned diluted mixed immunogen solution each time).

[0162] Fourteen days after the third immunization with the above single immunogens and mixed immunogens (i.e., day 56 of the entire procedure), mouse sera were collected and the titers of neutralizing antibodies induced by them were evaluated by pseudovirus neutralization experiments.

[0163] The neutralizing antibody titer test results of each immune mouse serum against SARS-CoV-2D614G mutant and pseudoviruses of each subtype of Omicron (including: BA.2, BQ.1.1, XBB, JN.1, KP.2, KP.3.1.1) are as follows Figure 8 As shown; Figure 8 The results showed that the trimeric immunogens XBB-BQ.1.1-SARS, XBB-JN.1-SARS, BQ.1.1-JN.1-SARS and the mixed immunogen composed of PT-XBB-SARS and BQ.1.1-JN.1-SARS were able to induce mice to produce high-titer neutralizing antibodies against various subtype variants of SARS-CoV-2Omicron, suggesting that they have a broad-spectrum immune protection effect against various subtype variants of SARS-CoV-2Omicron.

[0164] The neutralizing antibody titer test results of each immune mouse serum against SARS-CoV and BtSY2, Pangolin GD, WIV pseudovirus are as follows Figure 9 As shown; Figure 9 The results showed that the trimeric immunogens XBB-BQ.1.1-SARS, XBB-JN.1-SARS, BQ.1.1-JN.1-SARS, and the mixed immunogen composed of PT-XBB-SARS and BQ.1.1-JN.1-SARS were able to induce mice to produce high-titer neutralizing antibodies against SARS-CoV, BtSY2, Pangolin GD, and WIV. This indicates that the trimeric immunogens in this study can not only effectively increase the neutralizing antibody titers of mice against various mutant strains of SARS-CoV-2, but also have high neutralizing titers against strains of Sabei coronavirus. In other words, the trimeric immunogen design has a broad-spectrum protective effect against Sabei coronavirus strains.

[0165] A radar chart was made of the neutralizing antibody titers against each Sabae coronavirus induced in mice by the trimeric immunogens PT-XBB-SARS, XBB-BQ.1.1-SARS, XBB-JN.1-SARS, BQ.1.1-JN.1-SARS, the mixed immunogen composed of PT-XBB-SARS and BQ.1.1-JN.1-SARS, and the dimeric immunogens BA.2-SARS, PT-SARS, PT-PT, and SARS-SARS tested in the above examples. The results are shown in the figure. Figure 10 .

[0166] Depend on Figure 10 The results show that the dimer immunogens BA.2-SARS, PT-SARS, PT-PT, SARS-SARS (see Figure 10Compared with the dotted line part), the trimeric immunogens PT-XBB-SARS, XBB-BQ.1.1-SARS, XBB-JN.1-SARS, BQ.1.1-JN.1-SARS and the mixed immunogen composed of PT-XBB-SARS and BQ.1.1-JN.1-SARS of the present invention showed universal and significantly enhanced neutralizing activity in response to various subtype variants of SARS-CoV-2Omicron (especially the latest epidemic strains SARS-CoV-2Omicron BQ.1.1, XBB, JN.1, KP.2, KP.3.1.1 variants) and non-SARS-CoV-2 Sabei coronaviruses (including SARS-CoV, BtSY2, Pangolin GD, WIV), thus showing a broad-spectrum and efficient immune protection effect in the entire Sabei coronavirus genus, which has very important practical significance.

[0167] 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 various embodiments of the present invention.

Claims

1. A recombinant trimeric immunogen of Sabella coronavirus, characterized in that The recombinant trimeric immunogen comprises an amino acid sequence arranged in the format shown in formula (I): (AB)-C1-(AB)1-C2-(AB)2 (I) In formula (I): (AB) and (AB)1 each independently represent the S protein RBD domain of the novel coronavirus prototype strain or variant strain or its functionally active fragment, or a derivative sequence thereof; (AB)2 represents the S protein RBD domain of severe acute respiratory syndrome coronavirus or its functionally active fragment, or a derivative sequence thereof; 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 trimeric immunogen according to claim 1, characterized in that In formula (I): (AB) represents the RBD domain of the S protein of the novel coronavirus prototype strain or a functionally active fragment thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity thereto; preferably, (AB) represents the amino acid sequence as shown in SEQ ID NO: 1; (AB)1 represents the RBD domain of the S protein of the novel coronavirus Omicron variant XBB subtype or a functionally active fragment 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; preferably, (AB)1 represents the amino acid sequence as shown in SEQ ID NO: 2; (AB)2 represents the S protein RBD domain of severe acute respiratory syndrome coronavirus or a functionally active fragment 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; preferably, (AB)2 represents the amino acid sequence as shown in SEQ ID NO:3; Preferably, n=0, 1, 2 or 3; Preferably, the recombinant trimeric immunogen comprises the amino acid sequence shown in SEQ ID NO:

9.

3. The recombinant trimeric immunogen according to claim 1, characterized in that In formula (I): (AB) represents the RBD domain of the S protein of the novel coronavirus Omicron variant XBB subtype or a functionally active fragment 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; preferably, (AB) represents the amino acid sequence as shown in SEQ ID NO: 4; (AB)1 represents the S protein RBD domain of the novel coronavirus Omicron variant BQ.1.1 subtype or a functionally active fragment 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; preferably, (AB)1 represents the amino acid sequence as shown in SEQ ID NO:5; (AB)2 represents the S protein RBD domain of severe acute respiratory syndrome coronavirus or a functionally active fragment 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; preferably, (AB)2 represents the amino acid sequence as shown in SEQ ID NO:3; Preferably, n=0, 1, 2 or 3; Preferably, the recombinant trimeric immunogen comprises the amino acid sequence shown in SEQ ID NO:

10.

4. The recombinant trimeric immunogen according to claim 1, characterized in that In formula (I): (AB) represents the RBD domain of the S protein of the novel coronavirus Omicron variant XBB subtype or a functionally active fragment 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; preferably, (AB) represents the amino acid sequence as shown in SEQ ID NO: 4; (AB)1 represents the RBD domain of the S protein of the novel coronavirus Omicron variant JN.1 subtype or a functionally active fragment 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; preferably, (AB)1 represents the amino acid sequence as shown in SEQ ID NO:6; (AB)2 represents the S protein RBD domain of severe acute respiratory syndrome coronavirus or a functionally active fragment 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; preferably, (AB)2 represents the amino acid sequence as shown in SEQ ID NO:3; Preferably, n=0, 1, 2 or 3; Preferably, the recombinant trimeric immunogen comprises the amino acid sequence shown in SEQ ID NO:

11.

5. The recombinant trimeric immunogen according to claim 1, characterized in that In formula (I): (AB) represents the S protein RBD domain of the novel coronavirus Omicron variant BQ.1.1 subtype or a functionally active fragment 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; preferably, (AB) represents the amino acid sequence as shown in SEQ ID NO: 7; (AB)1 represents the RBD domain of the S protein of the novel coronavirus Omicron variant JN.1 subtype or a functionally active fragment 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; preferably, (AB)1 represents the amino acid sequence as shown in SEQ ID NO:6; (AB)2 represents the S protein RBD domain of severe acute respiratory syndrome coronavirus or a functionally active fragment 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; preferably, (AB)2 represents the amino acid sequence as shown in SEQ ID NO:3; Preferably, n=0, 1, 2 or 3; Preferably, the recombinant trimeric immunogen comprises the amino acid sequence shown in SEQ ID NO:

12.

6. The recombinant trimeric immunogen according to any one of claims 1 to 5, characterized in that The N-terminus of the recombinant trimeric immunogen further includes a signal peptide, and the signal peptide preferably has an amino acid sequence as shown in SEQ ID NO: 13; and / or, the C-terminus further includes a tag sequence, and the tag is preferably a His tag.

7. A polynucleotide encoding the recombinant trimeric immunogen according to any one of claims 1 to 6; Optionally, the polynucleotide is a DNA molecule, preferably, the DNA molecule comprises or consists of a DNA sequence as shown in one of SEQ ID NOs: 14-17; Optionally, the polynucleotide is an mRNA molecule; preferably, the mRNA molecule comprises or consists of an RNA sequence corresponding to a DNA sequence as shown in one of SEQ ID NOs: 14-17.

8. A nucleic acid construct, expression vector or host cell comprising the polynucleotide according to claim 7.

9. Use of the recombinant trimeric immunogen according to any one of claims 1 to 6, the polynucleotide according to claim 7, or the nucleic acid construct, expression vector or host cell according to claim 8 in the preparation of a medicament for preventing and / or treating Sabei coronavirus infection; Optionally, the drug is a vaccine; Optionally, the Sabae coronavirus is selected from the group consisting of: novel coronavirus, severe respiratory syndrome coronavirus and other SARS-related coronaviruses; Further optionally, the novel coronavirus is selected from: SARS-CoV-2 prototype strain, D614G mutant strain and Omicron lineages BA.2, BQ.1.1, XBB, JN.1, KP.2, KP.3.1.1; Further optionally, the other SARS-related coronaviruses are selected from: WIV1, BtSY2 and Pangolin GD.

10. An immunogenic composition comprising the recombinant trimeric immunogen according to any one of claims 1 to 6, the polynucleotide according to claim 7 or the nucleic acid construct, expression vector or host cell according to claim 8, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

11. The immunogenic composition according to claim 10, which is a Sabei coronavirus recombinant protein vaccine, comprising the recombinant trimeric immunogen according to any one of claims 1 to 6 and an adjuvant; Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant.

12. The immunogenic composition according to claim 10, which is a Sabei coronavirus DNA vaccine, comprising: (1) Eukaryotic expression vector; and (2) a DNA sequence encoding the recombinant trimeric immunogen according to any one of claims 1 to 6, constructed and incorporated into the eukaryotic expression vector; Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

13. The immunogenic composition according to claim 10, which is a Sabella coronavirus mRNA vaccine, comprising: (I) an mRNA sequence encoding the recombinant trimeric immunogen according to any one of claims 1 to 6; and (II) Lipid nanoparticles.

14. The immunogenic composition according to claim 10, which is a Sabei coronavirus-viral vector vaccine, comprising: (1) Viral backbone vector; and (2) a DNA sequence encoding the recombinant trimeric immunogen according to any one of claims 1 to 6, constructed into the viral backbone vector; Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.

15. The immunogenic composition according to claim 10, which is a Sabella coronavirus nanoparticle vaccine, wherein The recombinant trimeric immunogen according to any one of claims 1 to 6 is covalently linked to a self-assembling nanoparticle protein and self-assembled into nanoparticles, such that the recombinant trimeric immunogen is presented on the surface of the nanoparticles; Optionally, the carrier of the nanoparticle is ferritin; further optionally, the recombinant trimer immunogen is connected to ferritin via a linker, or is covalently linked via a SpyTag / SpyCatcher connection system.

16. The immunogenic composition according to any one of claims 10 to 15, 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, a liquid for external use, or an injectable preparation; further preferably, the injectable preparation is a push-in preparation.

17. A kit comprising the recombinant trimeric immunogen according to any one of claims 1 to 6, the polynucleotide according to claim 7, the nucleic acid construct, expression vector or host cell according to claim 8 and / or the immunogenic composition according to any one of claims 10 to 16.

18. The kit according to claim 17, characterized in that The kit comprises: (1) the recombinant trimeric immunogen according to claim 2, the polynucleotide encoding the same, and / or the nucleic acid construct, expression vector or host cell comprising the polynucleotide; and (2) The recombinant trimeric immunogen according to claim 5, the polynucleotide encoding the same, and / or the nucleic acid construct, expression vector or host cell comprising the polynucleotide.

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