WO2025040122A1 - Beta-coronavirus recombinant chimeric antigen, preparation method therefor and use thereof - Google Patents

Abstract

A beta-coronavirus (β-CoV) recombinant chimeric antigen, a preparation method therefor and a use thereof. The β-CoV recombinant chimeric antigen is a chimeric dimer antigen formed by linking an RBD fragment of S protein of a SARS-CoV-2 Omicron variant BA.1 subtype or BA.2 subtype and an RBD fragment of S protein of SARS-CoV in series, which can not only stimulate an organism to generate high-level neutralizing antibodies against a SARS-CoV-2 prototype strain and various variants and against SARS-CoV, but also stimulate the organism to generate high-level neutralizing antibodies against other SARS-related coronaviruses, so that the β-CoV recombinant chimeric antigen is expected to become an immunogen for broad-spectrum vaccines against β-CoVs, and has great clinical application prospects and industrial values.

Description

A beta coronavirus recombinant chimeric antigen, preparation method and application thereof

Cross-references

This application claims priority to the Chinese patent application filed on August 23, 2023, with application number 202311065664.1 and invention name “A recombinant chimeric antigen of beta coronavirus, its preparation method and application”, the entire contents of which are incorporated herein by reference.

Technical Field

The present application relates to the field of biomedicine, and specifically to a beta coronavirus recombinant chimeric antigen, its related products, preparation methods and applications.

Background Art

Coronaviruses belong to the Coronavirus family, which is divided into four coronavirus genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. The coronaviruses of the Alpha- and Betacoronavirus genera mainly infect mammals. SARS-CoV and SARS-CoV-2 belong to the Betacoronavirus genus and the Sarbecovirus subgenus. They are both positive-strand RNA viruses with envelopes. In addition, animal-derived SARS-related coronaviruses RaTG13, LYRa11, Pangolin GX, Pangolin GD, and WIV1 also belong to the Sarbecovirus subgenus. These viruses can widely infect humans and animals and are highly lethal. Therefore, it is of great significance to develop corresponding vaccines.

The surface spike protein (S protein) is the main neutralizing antigen of coronavirus. The receptor binding domain (RBD) of the S protein of SARS-CoV and SARS-CoV-2 is considered to be the most important antigenic target region for inducing the body to produce neutralizing antibodies. As a vaccine, RBD can focus the neutralizing antibodies stimulated by the body on the receptor binding to the virus, which can improve the immunogenicity and immune efficiency of the vaccine. It is currently believed that Sarbecovirus subgenus coronaviruses such as SARS-CoV and SARS-CoV-2 may enter cells by binding to the host cell receptor ACE2 through their RBD.

The information disclosed in this background technology section is only intended to increase the understanding of the overall background of the application and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

Summary of the invention

Purpose of the Invention

The purpose of this application is to provide a broad-spectrum and immunogenic β-coronavirus recombinant chimeric antigen, its related vaccine products, and its preparation method and application.

Solution

To achieve the purpose of this application, this application provides the following technical solutions:

In the first aspect, the present application provides a beta coronavirus recombinant chimeric antigen, wherein the amino acid sequence of the beta coronavirus recombinant chimeric antigen comprises, in order from N-terminus to C-terminus: an amino acid sequence arranged in the pattern of (A-B)-C-(A'-B'), wherein:

AB represents the RBD domain of the S protein of the novel coronavirus Omicron variant BA.1 subtype or BA.2 subtype or its an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity thereto,

A'-B' represents the amino acid sequence of the RBD domain of the S protein of severe respiratory syndrome coronavirus or a portion thereof, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity thereto, and,

C represents a linker (GGS)n, wherein n is an integer between 0 and 10.

For the above-mentioned beta coronavirus recombinant chimeric antigen:

Preferably, A-B represents an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, 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, A'-B' represents an amino acid sequence as in SEQ ID NO:4, 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, in the linker (GGS)n, n is an integer between 0 and 5, more preferably 0.

Further preferably, A-B represents an amino acid sequence such as SEQ ID NO: 1 or SEQ ID NO: 2; and/or, A'-B' represents an amino acid sequence such as SEQ ID NO: 4;

Further preferably, the β-coronavirus recombinant chimeric antigen has an amino acid sequence selected from the following: SEQ ID NO: 5, SEQ ID NO: 6.

In a feasible preferred embodiment, the N-terminus of the β coronavirus recombinant chimeric antigen further includes a signal peptide; preferably, the signal peptide has an amino acid sequence as shown in SEQ ID NO:7;

In a feasible preferred embodiment, the C-terminus of the β-coronavirus recombinant chimeric antigen also includes a histidine tag.

In a second aspect, the present application provides a method for preparing a recombinant chimeric antigen of a beta coronavirus as described in the first aspect above, comprising the following steps:

A sequence encoding a signal peptide is added to the 5' end of the nucleotide sequence encoding the recombinant chimeric antigen of the β coronavirus as described in the first aspect above, and a sequence encoding a histidine tag is added to the 3' end, and cloned and expressed, the correct recombinant is screened, and then transfected into cells of the expression system for expression. After expression, the cell supernatant is collected and purified to obtain the recombinant chimeric antigen of the β coronavirus.

For the above preparation method, preferably, the expression system cells are mammalian cells, insect cells, yeast cells or bacterial cells;

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

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

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

Optionally, the bacterial cells are Escherichia coli cells.

In a third aspect, the present application provides a polynucleotide encoding a recombinant chimeric antigen of a beta coronavirus as described in the first aspect above.

In specific embodiments, the polynucleotide is DNA or mRNA;

Preferably, the polynucleotide is a DNA sequence as shown in SEQ ID NO: 9 or 10;

Preferably, the polynucleotide is an mRNA sequence as shown in SEQ ID NO:11 or 12.

In a fourth aspect, the present application provides a nucleic acid construct, which comprises the polynucleotide as described in the third aspect above, and optionally, at least one expression control element operably linked to the polynucleotide.

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

In a sixth aspect, the present application provides a host cell, which is transformed or transfected with the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, or the expression vector as described in the fifth aspect.

In the seventh aspect, the present application provides the use of the recombinant chimeric antigen of the β-coronavirus as described in the first aspect, the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, the expression vector as described in the fifth aspect, or the host cell as described in the sixth aspect in the preparation of a drug for preventing and/or treating β-coronavirus infection.

Optionally, the drug is a vaccine;

Optionally, the beta coronavirus is a beta coronavirus of the Sarbecovirus subgenus, preferably selected from: novel coronavirus, severe respiratory syndrome coronavirus and other SARS-related coronaviruses;

Further preferably, the novel coronavirus includes: SARS-CoV-2 prototype 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 subtypes BA.1, BA.2, BA.2.75, BA.4/BA.5, BA.2.3.20, BQ.1.1, XBB, BF.7;

Further preferably, the other SARS-related coronaviruses include RaTG13, LYRa11, Pangolin GX, Pangolin GD, WIV1, preferably LYRa11 or WIV1.

In the eighth aspect, the present application provides a vaccine or immunogenic composition, which comprises the recombinant chimeric antigen of the beta coronavirus as described in the first aspect above, the polynucleotide as described in the third aspect above, the nucleic acid construct as described in the fourth aspect above, the expression vector as described in the fifth aspect above or the host cell as described in the sixth aspect above, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and/or diluent.

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

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

In another preferred embodiment, the vaccine or immunogenic composition is a beta coronavirus DNA vaccine comprising:

(1) a eukaryotic expression vector; and

(2) a DNA sequence encoding the beta coronavirus recombinant chimeric antigen as described in the first aspect above, which is constructed into the eukaryotic expression vector;

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

In another preferred embodiment, the vaccine or immunogenic composition is a beta coronavirus mRNA vaccine, and the mRNA vaccine comprises:

(I) an mRNA sequence encoding the beta coronavirus recombinant chimeric antigen as described in the first aspect above; and

(II) Lipid nanoparticles.

In another preferred embodiment, the vaccine or immunogenic composition is a beta coronavirus-viral vector vaccine, which comprises:

(1) viral backbone vectors; and

(2) a DNA sequence encoding the beta coronavirus recombinant chimeric antigen as described in the first aspect above, which is 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, adeno-associated virus vector.

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

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

Preferably, the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film-coated capsules, pellets, sublingual tablets and ointments;

Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid, an injectable or a pushable preparation.

In the ninth aspect, the present application provides a kit comprising the recombinant chimeric antigen of the beta coronavirus as described in the first aspect above, the polynucleotide as described in the third aspect above, the nucleic acid construct as described in the fourth aspect above, the expression vector as described in the fifth aspect above, the host cell as described in the sixth aspect above and/or the vaccine or immunogenic composition as described in the eighth aspect above, and optionally other types of beta coronavirus vaccines.

In a tenth aspect, the present application provides a method for preventing and/or treating a beta coronavirus infectious disease, the method comprising:

A preventive and/or therapeutically effective amount of the following substances is administered to a subject in need: a recombinant chimeric antigen of a beta coronavirus as described in the first aspect above, a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above, an expression vector as described in the fifth aspect above, a host cell as described in the sixth aspect above, and/or a vaccine or immunogenic composition as described in the eighth aspect above.

The "preventive and/or therapeutic effective amount" may vary depending on the subject, subject organ, symptoms, method of administration, etc. However, there are differences, which can be determined based on the doctor's judgment, taking into account the type of dosage form, method of administration, patient's age and weight, patient's symptoms, etc.

Beneficial Effects

The beta coronavirus recombinant chimeric antigen of the present application is a chimeric dimer antigen formed by the series connection of the S protein RBD fragment of the SARS-CoV-2 Omicron variant BA.1 subtype or BA.2 subtype and the S protein RBD fragment of SARS-CoV; when it is used to immunize mice, it can not only stimulate the body to produce high-level neutralizing antibodies against the SARS-CoV-2 prototype strain and each variant strain, and high-level neutralizing antibodies against SARS-CoV, but also stimulate the body to produce high-level neutralizing antibodies against other SARS-related coronaviruses (including RaTG13, LYRa11, Pangolin GX, Pangolin GD, WIV1, etc.); in particular, the level of neutralizing antibodies induced by it against SARS-CoV and other SARS-related coronaviruses (especially LYRa11 and WIV1) is significantly higher than that of the chimeric RBD dimer antigen of the SARS-CoV-2 prototype strain and SARS-CoV, achieving an unexpected technical effect, and is expected to become the immunogen of a broad-spectrum vaccine against beta coronaviruses.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIG1 is a diagram showing the molecular sieve chromatography curves of five recombinant proteins (BA.1-S, BA.2-S, CS, CC, SS) expressed by 293F cells and the SDS-PAGE identification results of the eluate at the elution peak.

Figure 2 shows the neutralizing antibody levels against the original strain (Prototype) and a series of variants (Delta, BA.1, BA.2, BA.2.75, BA.4/BA.5, BA.2.3.20, BQ.1.1, XBB, BF.7) of the new coronavirus in the serum of mice after three immunizations with five dimeric immunogens (BA.1-S, BA.2-S, CS, CC, SS) and the control group (PBS) detected by pseudovirus neutralization experiments; wherein, the horizontal axis shows the group (see Table 1 for grouping), and the vertical axis shows the neutralizing antibody titer (pVNT ₅₀ ).

FIG3 is a radar chart produced based on the results of FIG2 .

Figure 4 shows the neutralizing antibody levels against SARS-CoV in the serum of mice after three immunizations with five dimeric immunogens (BA.1-S, BA.2-S, CS, CC, SS) and a control group (PBS) detected by pseudovirus neutralization experiments; wherein the abscissa shows the groups (grouping is shown in Table 1), and the ordinate shows the neutralizing antibody titer (pVNT ₅₀ ).

Figure 5 shows the neutralizing antibody levels against SARS-related coronaviruses LYRa11 and WIV1 in the serum of mice after three immunizations of five dimeric immunogens (BA.1-S, BA.2-S, CS, CC, SS) and a control group (PBS) detected by pseudovirus neutralization experiments; wherein the abscissa shows the groups (grouping is shown in Table 1), and the ordinate shows the neutralizing antibody titer (pVNT ₅₀ ).

FIG6 shows the specific binding antibody titers against SARS-related coronavirus RmYN02 in the serum of mice immunized three times with five dimeric immunogens (BA.1-S, BA.2-S, CS, CC, SS) and a control group (PBS) detected by enzyme-linked immunosorbent assay (ELISA); wherein the horizontal axis shows the groups (see Table 1 for grouping). The ordinate shows the binding antibody titer.

DETAILED DESCRIPTION

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

In addition, in order to better illustrate the present application, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In certain 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 subject matter of the present application.

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.

Example 1: Design of the dimer antigens BA.1-SARS, BA.2-SARS and the control dimer antigens PT-SARS, PT-PT, SARS-SARS of the present application

In this embodiment, the dimeric antigen constructs BA.1-SARS (referred to as BA.1-S) and BA.2-SARS (referred to as BA.2-S) as representatives of the present application, and the dimeric antigen constructs PT-SARS (referred to as CS), PT-PT (referred to as CC), and SARS-SARS (referred to as SS) as controls were designed respectively. The specific scheme is as follows:

(1) Dimeric antigen BA.1-S

The sequence of the R319-K537 segment of the RBD domain of the SARS-CoV-2 Omicron variant BA.1 subtype (SEQ ID NO: 1) was directly connected in series with the sequence of the R306-Q523 segment of the RBD domain of the SARS-CoV (SEQ ID NO: 4), a signal peptide (MIHSVFLLMFLLTPTES, SEQ ID NO. 7) was connected to its N-terminus, and 6 histidines (HHHHHH) were added to its C-terminus to obtain the construct of the dimeric antigen BA.1-S, whose amino acid sequence is shown in SEQ ID NO: 13;

(2) Dimeric antigen BA.2-S

The sequence of the R319-K537 segment of the RBD domain of the SARS-CoV-2 Omicron variant BA.2 subtype (SEQ ID NO: 2) was directly connected in series with the sequence of the R306-Q523 segment of the RBD domain of the SARS-CoV (SEQ ID NO: 4), a signal peptide (MIHSVFLLMFLLTPTES, SEQ ID NO. 7) was connected to its N-terminus, and 6 histidines (HHHHHH) were added to its C-terminus to obtain the construct of the dimeric antigen BA.2-S, whose amino acid sequence is shown in SEQ ID NO: 14;

(3) Dimeric antigen CS

The sequence of the R319-K537 segment of the RBD domain of the SARS-CoV-2 prototype strain (SEQ ID NO: 3) and the sequence of the R306-Q523 segment of the RBD domain of the SARS-CoV (SEQ ID NO: 4) were directly connected in series. The end of the peptide was connected to a signal peptide (MIHSVFLLMFLLTPTES, SEQ ID NO.7), and six histidines (HHHHHH) were added to the C-terminus to obtain a construct of a dimer antigen CS, the amino acid sequence of which is shown in SEQ ID NO:15;

(4) Dimeric antigen CC

The sequences of the R319-K537 segment of the RBD domain of the two SARS-CoV-2 prototype strains (SEQ ID NO: 3) were directly connected in series, a signal peptide (MIHSVFLLMFLLTPTES, SEQ ID NO. 7) was connected to the N-terminus, and six histidines (HHHHHH) were added to the C-terminus to obtain the construct of the dimer antigen CC, whose amino acid sequence is shown in SEQ ID NO: 16;

(5) Dimeric antigen SS

The sequences of two segments of the R306-Q523 region of the SARS-CoV RBD domain (SEQ ID NO:4) were directly concatenated, a signal peptide (MFIFLLFLTLTSG, SEQ ID NO.8) was connected to the N-terminus, and six histidines (HHHHHH) were added to the C-terminus to obtain the construct of the dimeric antigen SS, whose amino acid sequence is shown in SEQ ID NO:17.

Example 2: Expression and purification of BA.1-S, BA.2-S, CS, CC, SS dimer antigen proteins

Construction of expression plasmid:

The amino acid sequences of the five constructs designed in the above Example 1 were optimized using human codons, and the corresponding DNA coding sequences are shown in SEQ ID NO: 18, 19, 20, 21, and 22, 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, and the resulting DNA sequences were synthesized by Anshengda Company; the five synthesized DNA sequences were cloned into the pCAGGS plasmid (commercially available) by double restriction digestion with EcoRI and Xhol, and expression plasmids pCAGGS-BA.1-S, pCAGGS-BA.2-S, pCAGGS-CS, pCAGGS-CC, and pCAGGS-SS expressing the above five dimeric antigen proteins were obtained, respectively.

Protein expression and purification:

1) The above-constructed expression plasmids pCAGGS-BA.1-S, pCAGGS-BA.2-S, pCAGGS-CS, pCAGGS-CC, and pCAGGS-SS 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.

2) purifying the obtained cell supernatant by nickel affinity column chromatography;

The conditions and procedures for nickel affinity column chromatography are as follows: HisTrap excel (cytiva) chromatography column with a column volume CV of 5 ml and a flow rate of 1-2 ml/min; pretreatment: first rinse with double distilled water for 5-10 CVs: equilibrium: rinse with Binding buffer (1×PBS buffer, pH7.4) for 5-10 CVs; loading: take 293F suspension cell culture filtered by 0.22 μm filter membrane and load it onto the balanced 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, pH7.4) until the UV baseline is flat, collect the eluted components 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.

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

The conditions and procedure of gel filtration chromatography are as follows:

A Superdex ^TM 200 Increase 10/300GL (cytiva) gel filtration chromatography column was used with a column volume CV of 24 ml and a flow rate of 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 proteins, and the target proteins can be combined and concentrated to obtain dimeric antigen proteins BA.1-S, BA.2-S, CC and SS.

A HiLoad ^TM 16/600 Superdex ^TM 200pg (cytiva) gel filtration chromatography column was used, with a column volume CV of 120ml and a flow rate of 1.0ml/min; pretreatment: rinse with double distilled water for 1 CV; equilibration: equilibrate with Binding buffer (1×PBS buffer, pH7.4) 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 dimeric protein CS.

The molecular sieve chromatography curves of the five dimeric antigen proteins BA.1-S, BA.2-S, CS, CC, and SS are shown in Figure 1. Figure 1 shows that BA.1-S, BA.2-S, CC, and SS all have a main elution peak when the elution volume is about 14 mL, and CS has a main elution peak when the elution volume is about 76 mL; the eluates at the above main elution peaks were collected for SDS-PAGE analysis, and the results showed that the eluted proteins at the main elution peaks were all around 60 KDa in size, which was consistent with the molecular size of the above five dimeric proteins, indicating that the five recombinant proteins all existed stably in the form of dimers, proving that the purified dimeric proteins of BA.1-S, BA.2-S, CS, CC, and SS were obtained; and the electrophoresis bands were single, indicating that the purified proteins had a high purity.

According to the above method, five purified dimeric antigen proteins BA.1-S, BA.2-S, CS, CC, and SS were obtained.

Example 3: Experimental animal immunization and sample collection

In order to detect the immunogenicity of the dimeric antigen protein, we used the five purified dimeric antigen proteins obtained in Example 2 as immunogens to immunize BALB/c mice.

Vaccine preparation

The dimer proteins BA.1-S, BA.2-S, CS, CC, and SS were diluted to 10 μg/ml with PBS, and the diluted immunogens were fully mixed and thoroughly emulsified with an MF59-like adjuvant, AddaVaxTM adjuvant, in a volume ratio of 1:1 to prepare vaccines. At the same time, the PBS solution was mixed with AddaVaxTM adjuvant as a negative control group.

Immunization regimen

The vaccine obtained according to the above method was used to immunize BALB/c mice (purchased from Vital River Company, female, 6-8 weeks old) by intramuscular injection. The experiment was divided into 6 groups, with 8 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 immunization was 100 μL (containing 1 μg of antigen protein).

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

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

The blood collected after each immunization was centrifuged at 12000 rpm for 10 min to collect the serum, which was incubated at 56°C for 30 min to inactivate complement and then stored at -80°C for subsequent experiments.

Example 4: Detection of neutralizing antibody titers in sera of immunized mice by pseudovirus neutralization experiment

In this example, the neutralizing antibody titers (pVNT 50 ) of the mouse sera after the third immunization (i.e., collected on the 56th day) were respectively detected against the prototype strain of the new coronavirus, the Delta variant, the Omicron variant BA.1, BA.2, BA.2.75, BA.4/ ₅ , BA.2.3.20, BQ.1, BQ.1.1, XBB, BF.7 subtypes, SARS-CoV, and pseudoviruses of SARS-related coronaviruses LYRa11 and WIV1.

The packaging of the novel coronavirus prototype strain and its various variants, SARS-CoV, LYRa11 and WIV1 pseudoviruses used in this example were 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 antisera to 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.

The method for detecting the titer of pseudovirus neutralizing antibodies against SARS-CoV and SARS-CoV-2 prototype strains and various variants is as follows:

In a 96-well plate, immune mouse serum was diluted in a 2-fold gradient with DMEM medium (containing 10% fetal bovine serum), with an initial dilution of 1:40, and a total of 10 gradients were set; similarly, pseudovirus was diluted to 2000TU/100μl with DMEM medium (containing 10% fetal bovine serum); then, the diluted immune mouse serum and diluted pseudovirus were mixed in a volume ratio of 1:1, blank medium mixed with pseudovirus was used as a negative control (NC), and blank medium not mixed with pseudovirus was used 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, incubated at 37°C for 15 hours, and the positive cell value was detected and calculated by CQ1 confocal cell imager (Yokogawa), and 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 _pVNT50 .

The method for detecting the titer of pseudovirus neutralizing antibodies against SARS-related coronaviruses LYRa11 and WIV1 is as follows:

In a 96-well plate, immune mouse serum was diluted in a 2-fold gradient with DMEM medium (containing 10% fetal bovine serum), with an initial dilution of 1:40, and a total of 10 gradients were set; similarly, pseudovirus was diluted to 1000TU/100μl with DMEM medium (containing 10% fetal bovine serum); then, the diluted immune mouse serum and the diluted pseudovirus were mixed in a volume ratio of 1:1, and the blank medium mixed with the pseudovirus was used as a negative control (NC), and the blank medium not mixed with the pseudovirus was used as a blank control (MOCK), and incubated at 37°C for 1 hour; then, the immune mouse serum was diluted in a 1:1 volume ratio with the pseudovirus, and the pseudovirus was mixed with the blank medium ... pseudovirus. The serum-pseudovirus mixture was transferred to a 96-well plate filled with 293T-ACE2 cells and incubated at 37°C for 15 h. The positive cells were detected and counted using a CQ1 confocal cell imager (Yokogawa). Then, a fitting curve was drawn in GraphPad Prism software to calculate the reciprocal of the serum dilution corresponding to 50% neutralization, which was the neutralization titer pVNT ₅₀ .

The neutralizing antibody titer detection results of each immunized mouse serum against the pseudovirus of the prototype strain of the new coronavirus and its variants are shown in Figure 2, and the corresponding radar chart is shown in Figure 3; the neutralizing antibody titer detection results of each immunized mouse serum against the pseudovirus of SARS-CoV are shown in Figure 4; the neutralizing antibody titer detection results of each immunized mouse serum against the pseudovirus of SARS-related coronaviruses LYRa11 and WIV1 are shown in Figure 5.

Figures 2 and 3 show that after immunizing BALB/c mice with the dimer antigens BA.1-S and BA.2-S of the present application as immunogens, they can induce high levels of neutralizing antibody titers against the prototype strain of the new coronavirus and its various types of variant pseudoviruses; in particular, the neutralizing antibody titers against various subtypes of pseudoviruses of the Omicron variant of the new coronavirus caused by BA.1-S and BA.2-S are significantly higher than those of the control dimer CS.

Figure 4 shows that after immunizing BALB/c mice with the dimer antigens BA.1-S and BA.2-S of the present application as immunogens, very high levels of neutralizing antibody titers against SARS-CoV pseudovirus can be induced, and the level of neutralizing antibodies against SARS-CoV pseudovirus induced by them is much higher than that of the control dimer CS, and is equivalent to that of the homozygous dimer SS; it should be noted that at the same immunization dose, compared with the homozygous dimer antigen SS, the actual content of SARS-CoV antigen components in the BA.1-S and BA.2-S dimer antigens is halved, however, the level of neutralizing antibodies against SARS-CoV pseudovirus induced by them is equivalent to that of SS; and compared with the chimeric dimer antigen CS of the SARS-CoV-2 prototype strain and SARS-CoV, the actual content of SARS-CoV antigen components in the BA.1-S and BA.2-S dimer antigens is equivalent, however, the level of neutralizing antibodies against SARS-CoV pseudovirus induced by them is much higher than that of CS (***, p<0.001). These results all indicate that the dimeric antigens BA.1-S and BA.2-S of the present application have achieved unexpected technical effects in eliciting neutralizing antibodies against SARS-CoV pseudovirus.

Figure 5 shows that after immunizing BALB/c mice with the dimer antigens BA.1-S and BA.2-S of the present application as immunogens, very high levels of neutralizing antibody titers against LYRa11 and WIV1 pseudoviruses can be induced, and the neutralizing antibody levels induced against LYRa11 and WIV1 pseudoviruses are significantly higher than those induced by the control dimer CS (for LYRa11 pseudovirus, ***, p<0.001; for WIV1 pseudovirus, *, p<0.05). These results show that the dimer antigens BA.1-S and BA.2-S of the present application have achieved unexpected technical effects in eliciting neutralizing antibodies against SARS-related coronaviruses LYRa11 and WIV1 pseudoviruses.

Example 5: Specific binding antibody titers against the related coronavirus RmYN02 in the sera of immunized mice by enzyme-linked immunosorbent assay (ELISA)

The RBD monomer protein of virus RmYN02 (obtained by artificial expression and purification according to the corresponding sequence displayed in the GenBank database) was diluted to 3ug/ml with ELISA coating solution (Solabo, C1050), and the diluted RmYN02 RBD monomer protein solution was added to the ELISA coating plate (Corning, 3590) at a volume of 100ul/well. ℃ overnight coating. After the overnight coating is completed, remove the coating solution containing antigen protein, add 5% skim milk prepared in PBS, add 100ul/well, and block at room temperature for 1 hour. During the blocking interval, dilute the mouse sera of different immune groups with 5% skim milk in a 4-fold gradient starting from 1:40. Specifically, in the first well, add 3.2ul mouse serum to 124.8ul skim milk, mix well, and complete the 1:40 dilution; take 32ul of liquid in the first well, mix it with 96ul skim milk, and complete the dilution of the second well; take 32ul of liquid in the second well, mix it with 96ul skim milk, and complete the dilution of the third well; and so on, in the dilution of the twelfth well, discard 32ul of liquid. After the blocking is completed, remove the skim milk, wash twice with PBST, drain the water, add gradient diluted immune mouse serum, 90ul per well, and incubate at 37℃ for 1.5 hours. After the incubation is completed, wash the plate 4 times with PBST and drain the water. At the same time, dilute the HRP-coupled secondary goat anti-mouse (Gene-Protein Link, P03S01M) with 5% skim milk at 1:3000, add 90ul to each well, incubate at 37°C for 1.5 hours. After the incubation is completed, rinse 4 times with PBST, drain the plate, and add TMB colorimetric solution (Biyuntian, P0209-500ml), 60ul per well. After the reaction time is appropriate, after the color development is completed, stop the color development with 2M HCl. Use an enzyme reader to detect the OD450 reading. A value greater than 2.5 times the blank control value is considered positive, and the antibody titer is the corresponding dilution multiple at this time; a result less than the minimum detection value of 1:40 is recorded as 1:20, i.e. 20. The results are shown in Figure 6.

Figure 6 shows that after immunizing BALB/c mice with the dimer antigens BA.1-S and BA.2-S of the present application as immunogens, very high levels of specific IgG antibody titers against RmYN02 pseudovirus can be induced, and the specific IgG antibody level against RmYN02 pseudovirus induced by them is significantly higher than that of the control dimer CS (*, p<0.05). This result shows that the dimer antigens BA.1-S and BA.2-S of the present application have achieved unexpected technical effects in eliciting specific binding antibodies against SARS-related coronavirus RmYN02 pseudovirus.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application 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 embodiments of the present application.

Industrial Applicability

The beta coronavirus recombinant chimeric antigen provided in the present application can not only stimulate the body to produce high-level neutralizing antibodies against the SARS-CoV-2 prototype strain and its variants, and high-level neutralizing antibodies against SARS-CoV, but also stimulate the body to produce high-level neutralizing antibodies against other SARS-related coronaviruses (including RaTG13, LYRa11, Pangolin GX, Pangolin GD, WIV1, etc.), and therefore is expected to become the immunogen for a broad-spectrum vaccine against beta coronaviruses, and has good clinical application value and industrialization prospects.

Claims (19)

A beta coronavirus recombinant chimeric antigen, characterized in that the amino acid sequence of the beta coronavirus recombinant chimeric antigen includes, in order from N-terminus to C-terminus: an amino acid sequence arranged in the pattern of (A-B)-C-(A'-B'), wherein: A-B represents the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Omicron variant BA.1 subtype or BA.2 subtype or a portion thereof, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and has the same or substantially the same immunogenicity thereto, A'-B' represents the amino acid sequence of the RBD domain of the S protein of severe respiratory syndrome coronavirus or a portion thereof, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity thereto, and, C represents a linker (GGS)n, wherein n is an integer between 0 and 10. The beta coronavirus recombinant chimeric antigen according to claim 1, characterized in that: A-B represents an amino acid sequence selected from the following: SEQ ID NO: 1, SEQ ID NO: 2, 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; and/or, A'-B' represents an amino acid sequence as in SEQ ID NO:4, 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; And/or, in the linker (GGS)n, n is an integer between 0 and 5. The beta coronavirus recombinant chimeric antigen according to claim 2, characterized in that: A-B represents an amino acid sequence such as SEQ ID NO: 1 or SEQ ID NO: 2; and/or, A'-B' represents an amino acid sequence such as SEQ ID NO: 4; Preferably, the β-coronavirus recombinant chimeric antigen has an amino acid sequence selected from the following: SEQ ID NO: 5, SEQ ID NO: 6. The beta coronavirus recombinant chimeric antigen according to any one of claims 1 to 3, characterized in that: the N-terminus of the beta coronavirus antigen also includes a signal peptide; preferably, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 7; And/or, the C-terminus of the β coronavirus antigen also includes a histidine tag. A method for preparing a beta coronavirus recombinant chimeric antigen as claimed in any one of claims 1 to 3, comprising the following steps: adding a sequence encoding a signal peptide to the 5' end of the nucleotide sequence encoding the beta coronavirus recombinant chimeric antigen as claimed in any one of claims 1 to 3, adding a sequence encoding a histidine tag to the 3' end, cloning and expressing, screening the correct recombinant, and then transfecting cells of the expression system for expression, collecting the cell supernatant after expression, and purifying to obtain the beta coronavirus recombinant chimeric antigen. The preparation method according to claim 5, characterized in that: the expression system cells are mammalian cells, insect cells, yeast cells or bacterial cells; Optionally, the mammalian cell is a HEK293T cell, a 293F series cell or a CHO cell; further Optionally, the 293F series cells are HEK293F cells, Freestyle293F cells or Expi293F cells; Optionally, the insect cell is a sf9 cell, a Hi5 cell, a sf21 cell or a S2 cell; Optionally, the yeast cell is a Pichia pastoris cell or a yeast cell transformed therefrom; Optionally, the bacterial cells are Escherichia coli cells. A polynucleotide encoding a recombinant chimeric beta coronavirus antigen as described in any one of claims 1 to 4. The polynucleotide according to claim 7, characterized in that: the polynucleotide is DNA or mRNA; Preferably, the polynucleotide is a DNA sequence as shown in SEQ ID NO: 9 or 10; Preferably, the polynucleotide is an mRNA sequence as shown in SEQ ID NO:11 or 12. A nucleic acid construct comprising the polynucleotide according to claim 7 or 8, and optionally, at least one expression control element operably linked to the polynucleotide. An expression vector comprising the nucleic acid construct according to claim 9. A host cell transformed or transfected with the polynucleotide according to claim 7 or 8, the nucleic acid construct according to claim 9 or the expression vector according to claim 10. Use of a beta coronavirus recombinant chimeric antigen according to any one of claims 1 to 4, a polynucleotide according to claim 7 or 8, a nucleic acid construct according to claim 9, an expression vector according to claim 10 or a host cell according to claim 11 in the preparation of a medicament for preventing and/or treating beta coronavirus infection; Optionally, the drug is a vaccine; Optionally, the beta coronavirus is a beta coronavirus of the Sarbecovirus subgenus, preferably selected from: novel coronavirus, severe respiratory syndrome coronavirus and other SARS-related coronaviruses; Further preferably, the novel coronavirus includes: SARS-CoV-2 prototype 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 subtypes BA.1, BA.2, BA.2.75, BA.4/BA.5, BA.2.3.20, BQ.1.1, XBB, BF.7; Further preferably, the other SARS-related coronaviruses include RaTG13, LYRa11, Pangolin GX, Pangolin GD, WIV1, preferably LYRa11 or WIV1. A vaccine or immunogenic composition comprising a beta coronavirus recombinant chimeric antigen as described in any one of claims 1 to 4, a polynucleotide as described in claim 7 or 8, a nucleic acid construct as described in claim 9, an expression vector as described in claim 10 or a host cell as described in claim 11, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and/or diluent. The vaccine or immunogenic composition according to claim 13, which is a beta coronavirus recombinant protein vaccine, comprising a beta coronavirus recombinant chimeric antigen and an adjuvant according to any one of claims 1 to 4; Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant. The vaccine or immunogenic composition according to claim 13, which is a beta coronavirus DNA vaccine, The DNA vaccines include: (1) a eukaryotic expression vector; and (2) a DNA sequence encoding a recombinant chimeric antigen of a beta coronavirus as described in any one of claims 1 to 4, which is constructed into the eukaryotic expression vector; Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors. The vaccine or immunogenic composition according to claim 13, which is a beta coronavirus mRNA vaccine, comprising: (I) an mRNA sequence encoding a beta coronavirus recombinant chimeric antigen as described in any one of claims 1 to 4; and (II) Lipid nanoparticles. The vaccine or immunogenic composition according to claim 13, which is a beta coronavirus-viral vector vaccine, comprising: (1) viral backbone vectors; and (2) a DNA sequence encoding a recombinant chimeric antigen of a beta coronavirus as described in any one of claims 1 to 4, which is 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, adeno-associated virus vector. The vaccine or immunogenic composition according to any one of claims 13 to 17, characterized in that the vaccine or immunogenic composition 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, powders, granules, fine granules, soft/hard capsules, film-coated capsules, pellets, sublingual tablets and ointments; Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid, an injectable or a pushable preparation. A kit comprising a beta coronavirus recombinant chimeric antigen as described in any one of claims 1 to 4, a polynucleotide as described in claim 7 or 8, a nucleic acid construct as described in claim 9, an expression vector as described in claim 10, a host cell as described in claim 11 and/or a vaccine or immunogenic composition as described in any one of claims 13 to 18, and optionally other types of beta coronavirus vaccines.