New coronavirus recombinant trimer antigen composition and application thereof

Abstract

The invention relates to a novel coronavirus immunogenic composition, a vaccine product based on the novel coronavirus immunogenic composition and application of the novel coronavirus immunogenic composition to preparation of a novel coronavirus vaccine. The novel coronavirus immunogenic composition can induce an organism to generate high-level neutralizing antibodies aiming at various recently appearing Omicron subtype variants such as BA.5, XBB, EG.5, BA.2. 86, JN.1, KP.2 and the like, and in the aspect, two recombinant chimeric antigens contained in the novel coronavirus immunogenic composition show an obvious synergistic effect, so that the novel coronavirus immunogenic composition has an excellent immune protection effect on the Omicron subtypes; besides, the immunogenic composition also shows relatively good immunogenicity for other new crown virus variants, so that the immunogenic composition is a broad-spectrum and effective new crown virus vaccine, can be used for coping with current complicated new crown virus epidemic situations, and has relatively high clinical application value and prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to a novel coronavirus immunogenic composition and its use in preparing a novel coronavirus vaccine. Background Art

[0002] The new coronavirus (SARS-CoV-2) belongs to the genus Beta-coronavirus of 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. Therefore, it is of great significance to develop a new generation of broad-spectrum and highly effective COVID-19 vaccines that can respond to new epidemic strains of the new coronavirus.

[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 area for inducing the body to produce neutralizing antibodies and is also an important target for the development of COVID-19 vaccines.

[0004] As SARS-CoV-2 evolved, a variety of epidemic variants emerged, including Alpha, Beta, Gamma, Delta and other strains. Omicron strains 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 neutralizing antibody drugs and vaccines for the new coronavirus. The need to develop a broad-spectrum and highly effective new coronavirus vaccine that can cope with a variety of epidemic variants is imminent, and the neutralization effect against Omicron and its new new coronavirus 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 new coronavirus immunogenic composition, a vaccine product based thereon, and its use in the preparation of a new coronavirus vaccine.

[0006] The novel coronavirus immunogenic composition of the present invention exhibits good immunogenicity against various variants of the novel coronavirus. In particular, it exhibits high-titer pseudovirus neutralization titers against the newly emerged Omicron subtypes BA.5, XBB, EG.5, BA.2.86, JN.1, KP.2, etc., and is therefore a broad-spectrum and effective novel coronavirus vaccine.

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

[0008] In a first aspect, the present invention provides an immunogenic composition, comprising:

[0009] (1) A recombinant chimeric antigen I composed of the RBD domain of the S protein of the Delta variant, the Omicron variant BA.1 subtype and the Omicron variant BA.2 subtype or its functionally active fragments or their derivative sequences in series, or its encoding nucleic acid;

[0010] (2) A recombinant chimeric antigen II composed of the S protein RBD domain of the 2019-nCoV Omicron variant BA.5 subtype, the Omicron variant XBB subtype and the Omicron variant BA.2.86 subtype or its functionally active fragments or their derivative sequences, or its encoding nucleic acid.

[0011] Preferably, the derivative sequence is an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the parent sequence and has the same or substantially the same immunogenicity as the parent sequence;

[0012] Preferably, the series connection is direct series connection or series connection via a joint; further preferably, the joint is (GGS) n ; Wherein, n=0,1,2,3,4 or 5.

[0013] In a preferred embodiment:

[0014] In the recombinant chimeric antigen I, the amino acid sequence of the S protein RBD domain of the novel coronavirus Delta variant or its functionally active fragment is as shown in SEQ ID NO: 1; and / or, the amino acid sequence of the S protein RBD domain of the novel coronavirus Omicron variant BA.1 subtype or its functionally active fragment is as shown in SEQ ID NO: 2; and / or, the amino acid sequence of the S protein RBD domain of the novel coronavirus Omicron variant BA.2 subtype or its functionally active fragment is as shown in SEQ ID NO: 3;

[0015] And / or, in the recombinant chimeric antigen II, the amino acid sequence of the S protein RBD domain of the BA.5 subtype of the new coronavirus Omicron variant or its functionally active fragment is as shown in SEQ ID NO:4; and / or, the amino acid sequence of the S protein RBD domain of the XBB subtype of the new coronavirus Omicron variant or its functionally active fragment is as shown in SEQ ID NO:5; and / or, the amino acid sequence of the S protein RBD domain of the BA.2.86 subtype of the new coronavirus Omicron variant or its functionally active fragment is as shown in SEQ ID NO:6.

[0016] In a further preferred embodiment:

[0017] The recombinant chimeric antigen I comprises the amino acid sequence shown in SEQ ID NO:7; preferably, the encoding nucleic acid of the recombinant chimeric antigen I comprises the DNA sequence shown in SEQ ID NO:8 or the RNA sequence shown in SEQ ID NO:9;

[0018] And / or, the recombinant chimeric antigen II comprises the amino acid sequence shown in SEQ ID NO:10; preferably, the encoding nucleic acid of the recombinant chimeric antigen II comprises the DNA sequence shown in SEQ ID NO:11 or the RNA sequence shown in SEQ ID NO:12.

[0019] For the recombinant chimeric antigen I and the recombinant chimeric antigen II, it is feasible that the N-terminus thereof further includes a signal peptide sequence, preferably, the signal peptide sequence is as shown in SEQ ID NO: 13; and / or, the C-terminus thereof further includes a tag sequence, preferably, the tag is a 6×His tag.

[0020] In a feasible specific implementation plan:

[0021] The recombinant chimeric antigen I comprises the amino acid sequence shown in SEQ ID NO: 14; preferably, the encoding nucleic acid of the recombinant chimeric antigen I comprises the DNA sequence shown in SEQ ID NO: 15 or the RNA sequence shown in SEQ ID NO: 16;

[0022] And / or, the recombinant chimeric antigen II comprises the amino acid sequence shown in SEQ ID NO:17; preferably, the encoding nucleic acid of the recombinant chimeric antigen II comprises the DNA sequence shown in SEQ ID NO:18 or the RNA sequence shown in SEQ ID NO:19.

[0023] In a second aspect, the present invention provides the use of the immunogenic composition described in the first aspect above in the preparation of a new coronavirus vaccine.

[0024] Optionally, the novel coronavirus vaccine is a preventive and / or therapeutic vaccine.

[0025] In a third aspect, the present invention provides a new coronavirus vaccine, which includes the immunogenic composition described in the first aspect above.

[0026] Optionally, the novel coronavirus vaccine also includes a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

[0027] In some preferred embodiments, the novel coronavirus vaccine is a novel coronavirus recombinant protein vaccine, which comprises the recombinant chimeric antigen I, the recombinant chimeric antigen II and an adjuvant;

[0028] 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.

[0029] In some other preferred embodiments, the novel coronavirus vaccine is a novel coronavirus DNA vaccine, and the DNA vaccine comprises:

[0030] (i) a eukaryotic expression vector; and

[0031] (ii) a DNA sequence encoding the recombinant chimeric antigen I and a DNA sequence encoding the recombinant chimeric antigen II constructed into the eukaryotic expression vector;

[0032] Preferably, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

[0033] In some other preferred embodiments, the novel coronavirus vaccine is a novel coronavirus mRNA vaccine, and the mRNA vaccine comprises:

[0034] (I) an mRNA sequence encoding the recombinant chimeric antigen I;

[0035] (II) an mRNA sequence encoding the recombinant chimeric antigen II; and

[0036] (III) carrier material;

[0037] Preferably, the carrier material is selected from the group consisting of lipid nanoparticles, protamine, polymer materials and inorganic nanoparticles, and more preferably lipid nanoparticles.

[0038] In some other preferred embodiments, the novel coronavirus vaccine is a novel coronavirus-viral vector vaccine, which comprises:

[0039] (1) viral backbone vectors; and

[0040] (2) constructing a DNA sequence encoding the recombinant chimeric antigen I and a DNA sequence encoding the recombinant chimeric antigen II into the viral backbone vector;

[0041] 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.

[0042] In other preferred embodiments, the novel coronavirus vaccine is a novel coronavirus nanoparticle vaccine;

[0043] Preferably, the carrier of the nanoparticle is ferritin, and the recombinant chimeric antigen I and the recombinant chimeric antigen II are covalently linked to ferritin and self-assembled into nanoparticles, so that the recombinant chimeric antigen I and the recombinant chimeric antigen II are presented on the surface of the nanoparticle; further preferably, the recombinant chimeric antigen I and the recombinant chimeric antigen II are connected to ferritin via a linker, or are covalently linked via a SpyTag / SpyCatcher connection system.

[0044] In a feasible embodiment, the novel coronavirus vaccine is in the form of a nasal spray, an oral preparation, a suppository or a parenteral preparation;

[0045] Preferably, the nasal spray is selected from aerosols, sprays and powder sprays;

[0046] Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film coatings and ointments;

[0047] Further preferably, the tablet is a sublingual tablet;

[0048] Further preferably, the granules are fine granules;

[0049] Further preferably, the powder is a powder;

[0050] Further preferably, the pills are pellets;

[0051] 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.

[0052] Fourthly, 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 immunogenic composition described in the first aspect above or the novel coronavirus vaccine described in the third aspect above.

[0053] 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.

[0054] 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.

[0055] Beneficial Effects

[0056] The novel coronavirus immunogenic composition provided by the present invention includes two recombinant chimeric antigens, which are respectively composed of the RBD domains or functionally active fragments of novel coronavirus Delta, Omicron BA.1 and Omicron BA.2, and the RBD domains or functionally active fragments of novel coronavirus OmicronBA.5, Omicron XBB and Omicron BA.2.86, thus covering a total of six serotypes of novel coronavirus.

[0057] The novel coronavirus immunogenic composition of the present invention can induce the body to produce high levels of neutralizing antibodies against the newly emerged Omicron subtype variants BA.5, XBB, EG.5, BA.2.86, JN.1, KP.2, etc. In this regard, the two recombinant chimeric antigens contained therein show obvious synergistic enhancement effects, and therefore, have excellent immune protection effects on these Omicron subtypes; in addition, the immunogenic composition also shows good immunogenicity for other novel coronavirus variants, and therefore, is a broad-spectrum and effective novel coronavirus vaccine that can be used to respond to the complex novel coronavirus epidemic and has high clinical application value and prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] 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.

[0059] Figure 1The recombinant expression plasmid pCAGGS-Delta-BA.1-BA.2 was transfected into Expi293F TM The molecular sieve chromatography curve of the recombinant trimeric antigen Delta-BA.1-BA.2 (abbreviated as D12) expressed later and the SDS-PAGE identification results of the eluate at its elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0060] Figure 2 The recombinant expression plasmid pCAGGS-BA.5-XBB-BA2.86 was transfected into Expi293F TM The molecular sieve chromatography curve of the recombinant trimeric antigen BA.5-XBB-BA2.86 (abbreviated as 5X6) expressed later and the SDS-PAGE identification results of the eluate at its elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0061] Figure 3 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.

[0062] Figure 4 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 the recombinant protein vaccines D12, 5X6, and D12+5X6 after one immunization by enzyme-linked immunosorbent assay (ELISA).

[0063] Figure 5 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 by mice stimulated by the recombinant protein vaccines D12, 5X6, and D12+5X6 after the second immunization by enzyme-linked immunosorbent assay (ELISA).

[0064] Figure 6 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 by mice stimulated by the recombinant protein vaccines D12, 5X6, and D12+5X6 after three immunizations by enzyme-linked immunosorbent assay (ELISA).

[0065] Figure 7 This is a graph of the neutralizing antibody titer test results against the D614G variant pseudovirus of the new coronavirus produced in the mouse serum after the third immunization of mice with the recombinant protein vaccines D12, 5X6, and D12+5X6 as recorded in Example 5 of the present invention.

[0066] Figure 8This is a graph of the neutralizing antibody titer test results against the Delta variant pseudovirus of the new coronavirus produced in the mouse serum after the third immunization of mice with the recombinant protein vaccines D12, 5X6, and D12+5X6 as recorded in Example 5 of the present invention.

[0067] Fig. 9 This is a graph showing the results of the neutralizing antibody titer test against the novel coronavirus Omicron BA.1 pseudovirus produced in the mouse serum by the recombinant protein vaccines D12, 5X6, and D12+5X6 after the third immunization of mice as recorded in Example 5 of the present invention.

[0068] Fig.10 This is a graph of the neutralizing antibody titer test results against the SARS-CoV-2 Omicron BA.2 pseudovirus produced in the mouse serum by the recombinant protein vaccines D12, 5X6, and D12+5X6 after the third immunization of mice as recorded in Example 5 of the present invention.

[0069] Fig.11 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 the recombinant protein vaccines D12, 5X6, and D12+5X6 as recorded in Example 5 of the present invention.

[0070] Fig.12 This is a graph of the results of the neutralizing antibody titer test against the SARS-CoV-2 Omicron XBB pseudovirus produced in the mouse serum after the third immunization of mice with the recombinant protein vaccines D12, 5X6, and D12+5X6 as recorded in Example 5 of the present invention.

[0071] Fig.13 This is a graph of the neutralizing antibody titer test results against the SARS-CoV-2 Omicron EG.5 pseudovirus produced in the mouse serum after the third immunization of mice with the recombinant protein vaccines D12, 5X6, and D12+5X6 as recorded in Example 5 of the present invention.

[0072] Fig.14 This is a graph of the neutralizing antibody titer test results against the SARS-CoV-2 Omicron BA.2.86 pseudovirus produced in the mouse serum after the third immunization of mice with the recombinant protein vaccines D12, 5X6, and D12+5X6 as recorded in Example 5 of the present invention.

[0073] Fig.15 This is a graph of the neutralizing antibody titer test results against the SARS-CoV-2 Omicron JN.1 pseudovirus produced in the mouse serum after the third immunization of mice with the recombinant protein vaccines D12, 5X6, and D12+5X6 as recorded in Example 5 of the present invention.

[0074] Fig.16 This is a graph of the neutralizing antibody titer test results against the SARS-CoV-2 Omicron KP.2 pseudovirus produced in the mouse serum after the third immunization of mice with the recombinant protein vaccines D12, 5X6, and D12+5X6 as recorded in Example 5 of the present invention.

[0075] Fig.17 It is a radar chart of the neutralizing antibody titer detection results against different strains of pseudoviruses produced in mouse serum after the third immunization of mice with the recombinant protein vaccines D12, 5X6, and D12+5X6 as recorded in Example 5 of the present invention. DETAILED DESCRIPTION

[0076] 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.

[0077] 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.

[0078] 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.

[0079] Example 1: Design of SARS-CoV-2 Delta-BA.1-BA.2 RBD trimer (abbreviated as D12), BA.5-XBB-BA.2.86 RBD trimer (abbreviated as 5X6) expression constructs

[0080] In this embodiment, expression constructs of the new coronavirus Delta-BA.1-BA.2RBD trimer (abbreviated as D12) and BA.5-XBB-BA.2.86RBD trimer (abbreviated as 5X6) were designed respectively, and the specific scheme is as follows:

[0081] (1) The sequence of the R319-K537 segment of the RBD domain of the Delta variant of the new coronavirus (SEQ ID NO: 1), the sequence of the V320-K537 segment of the RBD domain of the Omicron BA.1 variant (SEQ ID NO: 2), and the sequence of the V320-K537 segment of the RBD domain of the Omicron BA.2 variant (SEQ ID NO: 3) were directly connected in series, a signal peptide (SEQ ID NO: 13) was connected to the N-terminus, and a 6×His tag was added to the C-terminus to obtain the construct of the Delta-BA.1-BA.2 chimeric RBD trimer D12 (whose amino acid sequence is shown in SEQ ID NO: 14);

[0082] (2) The sequences of the R319-K537 segment of the RBD domain of the novel coronavirus Omicron BA.5 variant (SEQ ID NO: 4), the V320-K537 segment of the RBD domain of the Omicron XBB variant (SEQ ID NO: 5), and the V320-K537 segment of the RBD domain of the Omicron BA.2.86 variant (SEQ ID NO: 6) were directly connected in series, a signal peptide (SEQ ID NO: 13) was connected to the N-terminus, and a 6×His tag was added to the C-terminus to obtain a BA.5-XBB-BA.2.86 chimeric RBD trimer 5X6 construct (whose amino acid sequence is shown in SEQ ID NO: 17).

[0083] Example 2: Expression and purification of recombinant trimeric antigen proteins D12 and 5X6

[0084] Construction of recombinant expression plasmid:

[0085] The amino acid sequences of the expression constructs of the recombinant trimeric antigens D12 and 5X6 designed in the above Example 1 were optimized using human codons, and the corresponding DNA coding sequences were shown in SEQ ID NO:15 and SEQ ID NO:18, 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-Delta-BA.1-BA.2 and pCAGGS-BA.5-XBB-BA2.86 expressing the recombinant trimeric antigens D12 and 5X6, respectively.

[0086] Protein expression and purification:

[0087] Expi 293F cells were used to express single-chain heterologous RBD trimers of D12 and 5X6.

[0088] The recombinant expression plasmids pCAGGS-Delta-BA.1-BA.2 and pCAGGS-BA.5-XBB-BA2.86 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 (20mM Tris, 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, pH8.0, 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 TM 200 Increase 10 / 300GL column (GE Healthcare) was used for molecular sieve chromatography to further purify the target protein. The molecular sieve chromatography buffer was PBS buffer (8mM Na 2 HPO 4 , 136 mM NaCl, 2 mM KH 2 PO 4 , 2.6 mM KCl, pH 7.4).

[0089] The molecular sieve chromatography curves of the recombinant trimer antigens D12 and 5X6 and the SDS-PAGE identification results of the eluate at the elution peak are shown as follows: Figure 1 , Figure 2 shown by Figure 1 , 2It can be seen that D12 and 5X6 both have an elution peak at 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 two recombinant trimeric antigen proteins are stable in the form of disulfide-independent trimers, and also shows that the RBD trimeric proteins of D12 and 5X6 have indeed been purified, and the electrophoresis band is single, indicating that the purified protein has a high purity. In addition, the D12 and 5X6 RBD trimeric proteins also have a high yield.

[0090] Example 3: Experimental animal immunization and sample collection

[0091] In order to detect the immunogenicity of recombinant trimeric antigen D12, 5X6 and the mixed antigen D12+5X6 of the two, we used the purified trimeric antigen protein D12, 5X6 obtained in Example 2 and the mixed antigen D12+5X6 (1:1) of the two in a mass ratio of 1:1 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 Weitong Lihua Company, all female, 6-8 weeks old. The mouse grouping and immune dose situation are shown in Table 1.

[0092] Table 1. Grouping and immunization dose of mice immunized with the novel coronavirus RBD trimer vaccine

[0093] Grouping Immunogen Dosage (μg / mouse) pcs / group Adjuvant 1 PBS / 8 Addavax 2 D12 (Delta-BA.1-BA.2) 2 8 Addavax 3 5X6(BA.5-XBB-BA.2.86) 2 8 Addavax 4 D12+5X6(1:1) 2 8 Addavax

[0094] The specific experimental procedures are as follows:

[0095] The immunogens D12, 5X6 and D12+5X6 (1:1) 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.

[0096] Immunization regimen Figure 3As 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), and 50 μL was injected into each 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.

[0097] Example 4: Detection of antigen-specific antibody titers induced by recombinant trimeric antigen protein vaccines D12, 5X6 and D12+5X6 (1:1) by enzyme-linked immunosorbent assay (ELISA)

[0098] In this example, the antigen-specific antibody titer of the serum of mice immunized with the recombinant trimeric antigen protein vaccines D12, 5X6 and D12+5X6 (1:1) in Example 3 was detected by enzyme-linked immunosorbent assay (ELISA), and the specific procedure was as follows:

[0099] (1) D12, 5X6, and D12+5X6 trimer proteins were diluted to 3 μg / mL using ELISA coating solution (Solabo, C1050), and 100 μL of the dilution solution was added to each well of a 96-well ELISA plate (Coring, 3590). The plates were placed at 4°C overnight (more than 12 h) for coating.

[0100] (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;

[0101] (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 PBST;

[0102] (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.

[0103] 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.

[0104] The immunogenicity test results of the serum collected on day 19 (i.e., one immune serum) are as follows Figure 4 As shown; Figure 4 The results showed that D12, 5X6, and D12+5X6 trimer vaccines all produced corresponding antibodies after the first immunization. Specifically, the serum specific binding antibody titer of D12 trimer vaccine exceeded 10 4 The serum specific binding antibody titer after one immunization with 5X6 trimer vaccine was relatively low, less than 10 3 The serum specific binding antibody titer of D12+5X6 (1:1) vaccine after one immunization was 10 3 ~10 4 That is, the serum specific binding antibodies of the D12 immunization group (***) and the D12+5X6 immunization group (**) after immunization were significantly higher than those of the 5X6 immunization group.

[0105] The immunogenicity test results of the serum collected on day 35 (i.e., the second immunization serum) are as follows Figure 5 As shown; Figure 5 The results showed that the specific binding antibody titer induced by the D12+5X6 trimer vaccine group in mice after the second immunization was 10 6 The binding antibody of D12 is relatively high (more than 10 6 ), the binding antibody titer of 5X6 was lower (about 10 5 ), the specific binding antibody titers of D12 immunization group (**) and D12+5X6 immunization group (**) after the second immunization were significantly higher than those of 5X6 immunization group.

[0106] The immunogenicity test results of the serum collected on day 56 (i.e., triple-immune serum) are as follows: Figure 6 As shown; Figure 6 The results showed that the specific binding antibody titers induced by the D12, 5X6, and D12+5X6 trimer vaccine groups after the third immunization were all higher than 10 6 (In 10 6 -10 7 ), among which, the specific binding antibody of D12 immune group was the highest, followed by D12+5X6, and 5X6 was the lowest.

[0107] The above experimental results show that the three immunogens D12, 5X6 and D12+5X6 (1:1) can induce mice to produce high titers of antigen-specific binding antibodies. After the third immunization, the binding antibody titers were all higher than 10 6 .

[0108] Example 5: Neutralizing antibody titers produced by recombinant trimer antigen protein vaccines D12, 5X6 and D12+5X6 (1:1) against SARS-CoV-2 pseudoviruses were detected by pseudovirus neutralization experiments

[0109] 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 trimer antigen protein vaccines D12, 5X6 and D12+5X6 (1:1) in Example 3 was detected against the new coronavirus D614G variant, Delta variant and Omicron BA.1, BA.2, BA.5, XBB, BA.2.86, EG.5, JN.1 and KP.2 subtype pseudoviruses. 50 ).

[0110] 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).

[0111] The method for detecting the neutralizing antibody titer of the new coronavirus pseudovirus (hereinafter referred to as pseudovirus) is as follows:

[0112] 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 .

[0113] The results of the neutralizing antibody titers produced by the sera of mice in each immunization group after three immunizations against the D614G variant, 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 7-16 shown.

[0114] Figure 7-16 show:

[0115] 1) If Figure 7 Shown: Pseudovirus against the D614G variant of the new coronavirus, pVNT of serum from the D12 immune group 50 It is 24,332, pVNT of 5X6 immune group serum 50 20, pVNT of serum from D12+5X6 immune group 50 That is, for the pseudovirus of the D614G variant, the neutralizing effect of the D12+5X6 immunization group was much higher than that of the 5X6 immunization group, but slightly lower than that of the D12 immunization group.

[0116] 2) If Figure 8 Shown: Pseudovirus against the Delta variant of the new coronavirus, pVNT of serum from the D12 immune group 50 The pVNT of 29,641,5X6 immune group serum 50 13, pVNT of serum from D12+5X6 immune group 50 It was 6,015. That is, for the pseudovirus of the Delta variant, the neutralizing effect of the D12+5X6 immunization group was much higher than that of the 5X6 immunization group, but slightly lower than that of the D12 immunization group.

[0117] 3) If Fig. 9 Shown: Pseudovirus against the novel coronavirus Omicron BA.1 variant, pVNT of serum from the D12 immune group 50 The pVNT of 62,353,5X6 immune group serum 50 It is 385, pVNT of serum from D12+5X6 immune group 50 That is, for the pseudovirus of the Omicron BA.1 variant, the neutralizing effect of the D12+5X6 immunization group was much higher than that of the 5X6 immunization group, but slightly lower than that of the D12 immunization group.

[0118] 4) If Fig.10 Shown: Pseudovirus against the novel coronavirus Omicron BA.2 variant, pVNT of D12 immune group serum 50 is 45,378, while the pVNT of the 5X6 immune group serum 50 It is 435, pVNT of serum from D12+5X6 immune group 50That is, for the pseudovirus of the Omicron BA.2 variant, the neutralizing effect of the D12+5X6 immunization group was much higher than that of the 5X6 immunization group, but slightly lower than that of the D12 immunization group.

[0119] 5) If Fig.11 Shown: Pseudovirus against the novel coronavirus Omicron BA.5 variant, pVNT of serum from the D12 immune group 50 The pVNT of 5X6 immune group serum is 6,787 50 3,310, pVNT of serum from D12+5X6 immune group 50 That is, for the pseudovirus of the Omicron BA.5 variant, the neutralizing effect of the D12+5X6 immunization group was significantly higher than that of the 5X6 immunization group and the D12 immunization group (the antibody titer levels of the former were about 4.7 times and more than 2 times of the latter, respectively).

[0120] 6) If Fig.12 Shown: Pseudovirus against the novel coronavirus Omicron XBB variant, pVNT of serum from the D12 immune group 50 It is the pVNT of 34,5X6 immune group serum 50 It is 620, pVNT of serum from D12+5X6 immune group 50 The value was 2,152. That is, for the pseudovirus of the Omicron XBB variant, the neutralizing effect of the D12+5X6 immunization group was significantly higher than that of the 5X6 immunization group and the D12 immunization group (the antibody titer levels of the former were about 3.5 times and about 63.3 times of the latter, respectively).

[0121] 7) If Fig.13 Shown: Pseudovirus against the novel coronavirus Omicron EG.5 variant, pVNT of D12 immune group serum 50 is 40, while the pVNT of the 5X6 immune group serum 50 It is the pVNT of 401, D12+5X6 immune group serum 50 The neutralization effect of the D12+5X6 immunization group on the pseudovirus of the Omicron EG.5 variant was significantly higher than that of the 5X6 immunization group and the D12 immunization group (the antibody titer of the former was about 4.7 times and about 47.6 times that of the latter, respectively).

[0122] 8) If Fig.14 Shown: Pseudovirus against the novel coronavirus Omicron BA.2.86 variant, pVNT of D12 immune group serum 50 is 201, and the pVNT of the 5X6 immune group serum 50 It is 735, pVNT of serum from D12+5X6 immune group50 That is, for the pseudovirus of the Omicron BA.2.86 variant, the neutralizing effect of the D12+5X6 immunization group was significantly higher than that of the 5X6 immunization group and the D12 immunization group (the antibody titer levels of the former were about 4.1 times and about 15.2 times of the latter, respectively).

[0123] 9) If Fig.15 Shown: Pseudovirus against the novel coronavirus Omicron JN.1 variant, pVNT of D12 immune group serum 50 is 129, while the pVNT of the 5X6 immune group serum 50 It is the pVNT of 476, D12+5X6 immune group serum 50 It was 1,194. That is, for the pseudovirus of the Omicron JN.1 variant, the neutralizing effect of the D12+5X6 immunization group was significantly higher than that of the 5X6 immunization group and the D12 immunization group (the antibody titer levels of the former were about 2.5 times and about 9.3 times of the latter, respectively).

[0124] 10) As Fig.16 Shown: Pseudovirus against the novel coronavirus Omicron KP.2 variant, pVNT of D12 immune group serum 50 is 87, while the pVNT of the 5X6 immune group serum 50 It is the pVNT of 442, D12+5X6 immune group serum 50 The neutralization effect of the D12+5X6 immunization group on the pseudovirus of the Omicron KP.2 variant was significantly higher than that of the 5X6 immunization group and the D12 immunization group (the antibody titer of the former was about 2.5 times and about 12.6 times that of the latter, respectively).

[0125] Depend on Figure 7-16 The results show that for Omicron BA.5, XBB, EG.5, BA.2.86, JN.1 and KP.2 subtypes, the neutralizing effect of the D12 and 5X6 mixed vaccine immunization group at the same immunization dose was significantly higher than that of the D12 and 5X6 single immunization group, that is, in terms of inducing neutralizing antibodies against Omicron BA.5, XBB, EG.5, BA.2.86, JN.1 and KP.2 subtypes, D12 and 5X6 showed synergistic effects, and the mixed vaccine composed of the two can induce high levels of neutralizing protective antibodies. In addition, for other variants of the new coronavirus, they also showed good neutralizing effects.

[0126] 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.17 shown by Fig.17 It can be seen that:

[0127] Compared with the D12 immunization group and the 5X6 immunization group, the mouse serum of the D12+5X6 mixed immunization group showed a relatively balanced pseudovirus neutralization protection effect against various types of variants, whether it was the D614G variant, the Delta variant, or the Omicron BA.1, BA.2, BA.5, XBB, EG.5, BA.2.86, JN.1 and KP.2 variants, all maintained a high pseudovirus neutralization titer (1000-10000). In particular, for the newly emerged Omicron XBB, EG.5, BA.2.86, JN.1 and KP.2 subtypes, although the 5X6 single immunization group still had some pseudovirus neutralization protection effect, the neutralization titer was low (100-1000), while the D12 single immunization group almost lost the neutralization protection effect (10-201), while the D12+5X6 mixed immunization group still had a high neutralization protection effect.

[0128] The above experiments confirmed that the immunogenic composition of the present invention (i.e., the D12+5X6 immune group) not only has high immunogenicity against the D614G variant, the Delta variant, and the early Omicron variant subtypes BA.1, BA.2, and BA.5, but also can induce strong immune responses against the later Omicron variant subtypes XBB, EG.5, BA.2.86, JN.1, and KP.2 variants. This indicates that it has a balanced and effective immune protection effect against various variants of the new coronavirus, and has broad application prospects.

[0129] 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.

[0130] The sequences used in this article are as follows:

[0131] SEQ ID NO:1-Delta variant S protein RBD region R319-K537 amino acid sequence-219 aa

[0132] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0133] SEQ ID NO:2 - Amino acid sequence of the V320-K537 region of the RBD of the S protein of the Omicron BA.1 variant -218 aa

[0134] VQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0135] SEQ ID NO:3 - Amino acid sequence of the V320-K537 region of the RBD of the S protein of the Omicron BA.2 variant -218 aa

[0136] VQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0137] SEQ ID NO:4 - Amino acid sequence of the R319-K537 region of the S protein RBD of the Omicron BA.5 variant -219 aa

[0138] RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGSNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0139] SEQ ID NO:5 - Amino acid sequence of the V320-K537 region of the RBD of the S protein of the Omicron XBB variant -218 aa

[0140] VQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0141] SEQ ID NO:6 - amino acid sequence of the V320-K536 region of the S protein RBD of the Omicron BA.2.86 variant - 217 aa

[0142] VQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRLFRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0143] SEQ ID NO:7—Amino acid sequence of the recombinant trimeric antigen Delta-BA.1-BA.2 (ie, D12) (ie, SEQ ID NO: ID NO:1+SEQ ID NO:2+SEQ ID NO:3)-655 aa

[0144] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0145] SEQ ID NO:8 - DNA sequence encoding SEQ ID NO:7 - 1965 nt

[0146]

[0147] SEQ ID NO:9 - mRNA sequence encoding SEQ ID NO:7 - 1965 nt

[0148]

[0149] SEQ ID NO: 10 - Amino acid sequence of the recombinant trimeric antigen BA.5-XBB-BA2.86 (ie, 5X6) (ie, SEQ ID NO:4+SEQ ID NO:5+SEQ ID NO:6)-654 aa

[0150] RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGSNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKVQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRLFRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0151] SEQ ID NO:11 – DNA sequence encoding SEQ ID NO:10 – 1962 nt

[0152]

[0153] SEQ ID NO:12 – mRNA sequence encoding SEQ ID NO:10 – 1962 nt

[0154]

[0155] SEQ ID NO:13—Signal peptide-17aa

[0156] MIHSVFLLMFLLTPTES

[0157] SEQ ID NO: 14 - Complete construct sequence of the Delta-BA.1-BA.2 (D12) vaccine example (SEQ ID NO:7 +SEQ ID NO:1+SEQ ID NO:2+SEQ ID NO:3+ SEQ ID NO:8)- 678 aa

[0158] MIHSVFLLMFLLTPTESRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKHHHHHH*

[0159] SEQ ID NO:15 - DNA sequence encoding SEQ ID NO:14 Delta-BA.1-BA.2 (D12) construct - 2034 nt

[0160]

[0161] SEQ ID NO:16 - RNA sequence encoding SEQ ID NO:14 Delta-BA.1-BA.2 (D12) construct - 2034 nt

[0162]

[0163] SEQ ID NO: 17 - Complete construct sequence of BA.5-XBB-BA.2.86 (5X6) vaccine example (SEQ ID NO: 7+SEQ ID NO:4+SEQ ID NO:5+SEQ ID NO:6+SEQ ID NO:8)-677 aa

[0164] MIHSVFLLMFLLTPTESRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGSNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKVQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRLFRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKHHHHHH*

[0165] SEQ ID NO:18 - DNA encoding SEQ ID NO:17 BA.5-XBB-BA.2.86 (5X6) RBD trimer construct Sequence - 2031 nt

[0166]

[0167] SEQ ID NO:19 - RNA encoding SEQ ID NO:17 BA.5-XBB-BA.2.86 (5X6) RBD trimer construct Sequence - 2031 nt

[0168]

Claims

1. An immunogenic composition comprising: (1) A recombinant chimeric antigen I composed of the RBD domain of the S protein of the Delta variant, the Omicron variant BA.1 subtype and the Omicron variant BA.2 subtype or its functionally active fragments or their derivative sequences in series, or its encoding nucleic acid; (2) A recombinant chimeric antigen II composed of the S protein RBD domain of the 2019-nCoV Omicron variant BA.5 subtype, the Omicron variant XBB subtype and the Omicron variant BA.2.86 subtype or its functionally active fragments or their derivative sequences, or its encoding nucleic acid.

2. The immunogenic composition according to claim 1, characterized in that The derivative sequence is an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the parent sequence and has the same or substantially the same immunogenicity as the parent sequence; And / or, the series connection is direct series connection or series connection through a joint; preferably, the joint is (GGS) n ; Wherein, n=0,1,2,3,4 or 5.

3. The immunogenic composition according to claim 1, characterized in that In the recombinant chimeric antigen I, the amino acid sequence of the S protein RBD domain of the novel coronavirus Delta variant or its functionally active fragment is as shown in SEQ ID NO: 1; and / or, the amino acid sequence of the S protein RBD domain of the novel coronavirus Omicron variant BA.1 subtype or its functionally active fragment is as shown in SEQ ID NO: 2; and / or, the amino acid sequence of the S protein RBD domain of the novel coronavirus Omicron variant BA.2 subtype or its functionally active fragment is as shown in SEQ ID NO: 3; And / or, in the recombinant chimeric antigen II, the amino acid sequence of the S protein RBD domain of the BA.5 subtype of the new coronavirus Omicron variant or its functionally active fragment is as shown in SEQ ID NO:4; and / or, the amino acid sequence of the S protein RBD domain of the XBB subtype of the new coronavirus Omicron variant or its functionally active fragment is as shown in SEQ ID NO:5; and / or, the amino acid sequence of the S protein RBD domain of the BA.2.86 subtype of the new coronavirus Omicron variant or its functionally active fragment is as shown in SEQ ID NO:

6.

4. The immunogenic composition according to claim 3, characterized in that The recombinant chimeric antigen I comprises an amino acid sequence as shown in SEQ ID NO: 7; preferably, the encoding nucleic acid of the recombinant chimeric antigen I comprises a DNA sequence as shown in SEQ ID NO: 8 or an RNA sequence as shown in SEQ ID NO: 9; And / or, the recombinant chimeric antigen II comprises the amino acid sequence shown in SEQ ID NO: 10; preferably, the encoding nucleic acid of the recombinant chimeric antigen II comprises the DNA sequence shown in SEQ ID NO: 11 or the RNA sequence shown in SEQ ID NO:

12.

5. The immunogenic composition according to claim 3, characterized in that For the recombinant chimeric antigen I and the recombinant chimeric antigen II, their N-terminus also includes a signal peptide sequence, preferably, the signal peptide sequence is as shown in SEQ ID NO: 13; and / or, their C-terminus also includes a tag sequence, preferably, the tag is a 6×His tag.

6. The immunogenic composition according to claim 5, characterized in that The recombinant chimeric antigen I comprises an amino acid sequence as shown in SEQ ID NO: 14; preferably, the encoding nucleic acid of the recombinant chimeric antigen I comprises a DNA sequence as shown in SEQ ID NO: 15 or an RNA sequence as shown in SEQ ID NO: 16; And / or, the recombinant chimeric antigen II comprises the amino acid sequence shown in SEQ ID NO: 17; preferably, the encoding nucleic acid of the recombinant chimeric antigen II comprises the DNA sequence shown in SEQ ID NO: 18 or the RNA sequence shown in SEQ ID NO:

19.

7. Use of the immunogenic composition as described in any one of claims 1 to 6 in the preparation of a new coronavirus vaccine.

8. A novel coronavirus vaccine, characterized in that: The new coronavirus vaccine comprises the immunogenic composition as described in any one of claims 1-6.

9. The novel coronavirus vaccine according to claim 8, characterized in that The novel coronavirus vaccine is a novel coronavirus recombinant protein vaccine, which includes the recombinant chimeric antigen I, the recombinant chimeric antigen II 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.

10. The novel coronavirus vaccine according to claim 8, characterized in that The novel coronavirus vaccine is a novel coronavirus DNA vaccine, and the DNA vaccine comprises: (i) a eukaryotic expression vector; and (ii) a DNA sequence encoding the recombinant chimeric antigen I and a DNA sequence encoding the recombinant chimeric antigen II constructed into the eukaryotic expression vector; Preferably, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

11. The novel coronavirus vaccine according to claim 8, characterized in that The novel coronavirus vaccine is a novel coronavirus mRNA vaccine, and the mRNA vaccine comprises: (I) an mRNA sequence encoding the recombinant chimeric antigen I; (II) an mRNA sequence encoding the recombinant chimeric antigen II; and (III) carrier material; Preferably, the carrier material is selected from the group consisting of lipid nanoparticles, protamine, polymer materials and inorganic nanoparticles, preferably lipid nanoparticles.

12. The novel coronavirus vaccine according to claim 8, characterized in that The novel coronavirus vaccine is a novel coronavirus-viral vector vaccine, which includes: (1) viral backbone vectors; and (2) constructing a DNA sequence encoding the recombinant chimeric antigen I and a DNA sequence encoding the recombinant chimeric antigen II 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, adeno-associated virus vector.

13. The novel coronavirus vaccine according to claim 8, characterized in that The novel coronavirus vaccine is a novel coronavirus nanoparticle vaccine; Preferably, the carrier of the nanoparticle is ferritin, and the recombinant chimeric antigen I and the recombinant chimeric antigen II are covalently linked to ferritin and self-assembled into nanoparticles, so that the recombinant chimeric antigen I and the recombinant chimeric antigen II are presented on the surface of the nanoparticle; further preferably, the recombinant chimeric antigen I and the recombinant chimeric antigen II are connected to ferritin via a linker, or are covalently linked via a SpyTag / SpyCatcher connection system.

14. The novel coronavirus vaccine according to any one of claims 8 to 13, characterized in that The novel coronavirus vaccine is in the form of a nasal spray, an oral preparation, a suppository or a parenteral preparation; 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.

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