Broad-spectrum coronavirus protein chimeric nanoparticle vaccine as well as preparation method and application thereof

Abstract

The invention discloses a broad-spectrum coronavirus protein chimeric nanoparticle vaccine as well as a preparation method and application thereof. The coronavirus protein chimeric nanoparticle vaccine provided by the invention is a protein nanoparticle formed by self-assembly of a glycosylated and modified coronavirus RBD sequence, a glycosylated and modified coronavirus HRC, a glycosylated and modified DN5B sequence and a glycosylated and modified DN5A sequence. The vaccine can induce an organism to generate broad-spectrum immunoreactions aiming at various coronaviruses and variants thereof, so that a new solution is provided for dealing with spreading, prevention and control of the coronaviruses, and a technical foundation is laid for prevention of new variants of the coronaviruses possibly occurring in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of virus vaccines, and in particular to a broad-spectrum coronavirus protein chimeric nanoparticle vaccine and a preparation method and application thereof. Background Art

[0002] Coronaviruses are a class of enveloped, single-stranded, positive-sense RNA viruses encompassing four genera: α, β, γ, and δ. Many types of coronaviruses exhibit high interspecies transfer and can infect a wide range of species, including humans. They are highly contagious zoonotic pathogens. Since the 20th century, coronaviruses have repeatedly triggered global public health crises, including severe acute respiratory syndrome (SARS) in 2002 and Middle East respiratory syndrome (MERS) in 2012. These viral crises not only pose a serious threat to global health but also have profound socioeconomic impacts.

[0003] The genome of the coronavirus is extremely prone to mutation, which causes its surface proteins (such as the spike protein) to mutate frequently, resulting in the production of multiple variant viruses. This high variability makes it difficult for traditional vaccines targeting a single virus strain to cope with the constantly emerging virus variants, thereby reducing the vaccine's broad spectrum and long-term effectiveness. The rapid spread and continuous mutation of SARS-CoV-2 (such as the generation of Alpha, Beta, Delta, Omicron and other variants) have led to a decline in the protective efficacy of traditional vaccines, further exacerbating the difficulty of global epidemic prevention and control. Therefore, the development of a broad-spectrum vaccine that can cope with multiple coronaviruses and their variants has become an urgent need for current research.

[0004] Currently, vaccine development for coronaviruses is primarily focused on the spike protein (S) or its receptor-recognition domain (RBD). When the virus infects cells, the RBD of the S protein's S1 subunit first binds to the viral receptor on the target cell membrane. Subsequently, the S2 subunit undergoes a conformational change, and its N-terminal fusion peptide (FP) inserts into the target cell membrane. The S protein's S2 subunit contains two heptapeptide repeat domains (HRs), HR1 and HR2. HR1 and HR2 interact through residues to form a six-helix bundle (6-HB) core structure, shortening the distance between the viral and cellular membranes and enabling membrane fusion. As the core functional domain of the spike protein, the RBD possesses high immunogenicity, inducing the production of neutralizing antibodies that block viral binding to host cells. However, conventional RBD monomers exhibit weak immunogenicity, and single-strain RBD vaccines often struggle to address the highly variable nature of coronaviruses. Therefore, designing a vaccine with significantly enhanced immunogenicity and broad-spectrum protection has become a key research focus. Summary of the Invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide a broad-spectrum coronavirus protein chimeric nanoparticle vaccine and its preparation method and application. The present invention designs and prepares a broad-spectrum chimeric nanoparticle vaccine based on coronavirus protein, which forms an RBD-HRC trimer by forming a glycosylated RBD with an HRC domain, and chimerizes the RBD-HRC trimers of various coronaviruses and variants, and combines them with the I53 nanoparticle delivery system. It can induce the body to produce a broad-spectrum immune response against a variety of coronaviruses and their variants, thereby providing a new solution for responding to the spread and prevention and control of coronaviruses, and also laying a technical foundation for the prevention of new coronavirus variants that may appear in the future.

[0006] The first aspect of the present invention provides a fusion protein comprising: a glycosylated coronavirus RBD sequence, a coronavirus HRC and a DN5B sequence.

[0007] In the present invention, the term "coronavirus HRC" refers to an amino acid sequence starting from the first amino acid of HR1 (heptad repeat sequence 1) on the coronavirus genome and ending with the last amino acid of HR2 (heptad repeat sequence 2), that is, including the connecting part of HR1 and HR2).

[0008] In some embodiments of the present invention, the novel coronavirus includes at least one of SARS-CoV-2, SARS, MERS wild type and their variants.

[0009] In some embodiments of the present invention, the variant strain includes but is not limited to SARS-CoV-2-Omicron-BA.5.

[0010] In some embodiments of the present invention, the fusion protein consists of a glycosylated-modified coronavirus RBD sequence, a coronavirus HRC and a DN5B sequence connected in sequence.

[0011] In the present invention, the term "DN5B sequence" refers to the DN5B sequence of the I53 nanoparticle. The I53 nanoparticle is a computer-aided-designed two-component protein complex, formed by in vitro co-assembly of an icosahedral trimer (I53-50A, i.e., DN5A) and a dodecahedral pentamer (I53-50B, i.e., DN5B). It exhibits high symmetry and programmability. In some embodiments of the present invention, the sequence of DN5A is shown in SEQ ID NO: 5, and the sequence of DN5B is shown in SEQ ID NO: 6.

[0012] In some embodiments of the present invention, the coronavirus HRC is an amino acid sequence of the wild type SARS-CoV-2 starting from the first amino acid of HR1 (heptapeptide repeat sequence 1) on the coronavirus genome and ending with the last amino acid of HR2 (heptapeptide repeat sequence 2).

[0013] In some embodiments of the present invention, the glycosylation modification comprises at least one of the following:

[0014] Glycosylation occurs at R39 and / or P203 of the wild-type SARS-CoV-2 RBD sequence;

[0015] Glycosylation occurs at K28 and / or P191 positions of the SARS wild-type RBD sequence;

[0016] Glycosylation modification occurs at position V37 and / or T213 of the MERS wild-type RBD sequence; and

[0017] Glycosylation modification occurs at the R39 and / or P203 positions of the RBD sequence of the SARS-CoV-2 variant Omicron-BA.5.

[0018] In some embodiments of the present invention, the glycosylation modification is:

[0019] R39N and P203N glycosylation modifications occurred in the RBD sequence of the wild type of SARS-CoV-2;

[0020] K28N and P191N glycosylation modifications occurred in the RBD sequence of the SARS wild type;

[0021] V37N and T213N glycosylation modifications occur in the RBD sequence of the MERS wild type; and

[0022] R39N and P203N glycosylation modifications occurred in the RBD sequence of the SARS-CoV-2 variant Omicron-BA.5.

[0023] In some embodiments of the present invention, the fusion protein can be a single chimeric fusion protein (i.e., only a single glycosylated coronavirus RBD is chimeric) or a multi-chimeric fusion protein (i.e., multiple glycosylated coronavirus RBDs are chimeric at the same time).

[0024] In some embodiments of the present invention, the fusion protein comprises glycosylation-modified RBD sequences of at least two coronaviruses.

[0025] In some embodiments of the present invention, the fusion protein contains glycosylation-modified RBD sequences of two or three coronaviruses.

[0026] In some embodiments of the present invention, the fusion protein contains the glycosylation-modified RBD sequence of the wild type of SARS-CoV-2 and the glycosylation-modified RBD sequence of the SARS-CoV-2 variant strain Omicron-BA.5.

[0027] In some embodiments of the present invention, the fusion protein contains a glycosylated RBD sequence of the wild type of SARS-CoV-2, a glycosylated RBD sequence of the wild type of SARS, and a glycosylated RBD sequence of the wild type of MERS.

[0028] In some embodiments of the present invention, the fusion protein further comprises other functional groups.

[0029] In some embodiments of the present invention, the functional group includes at least one of a linker, a reporter group, a protein tag, a carrier, and a lipid group.

[0030] In the present invention, the position of the functional group is not limited. Based on actual use requirements and the specific selection of the functional group, it can be optionally inserted at any position at either end or in the middle of the fusion protein, provided that at least one function of the fusion protein is significantly reduced. In the present invention, the function includes immunogenicity.

[0031] In some embodiments of the present invention, the protein tag includes but is not limited to: affinity tags, such as GST, His, MBP, etc.; epitope tags, such as HA, Myc, Flag, etc.; fluorescent tags, such as GFP, mCherry, etc.; and other tags, such as SNAP and Avi, etc.

[0032] In some embodiments of the present invention, the carrier includes but is not limited to: carrier protein, such as hemocyanin, serum albumin, ovalbumin, etc.

[0033] In some embodiments of the invention, the lipid groups include, but are not limited to, fatty acid chains (such as palmitic acid) to form lipopeptides.

[0034] In some embodiments of the present invention, the fusion protein comprises:

[0035] (1) having a sequence as shown in any one of SEQ ID NOs: 1-4;

[0036] (2) Sequences that have one or more amino acids substituted and / or deleted and / or added to SEQ ID NOs: 1-4 and have the same functions as the sequences shown in SEQ ID NOs: 1-4;

[0037] (3) A sequence that is at least 90% identical to SEQ ID NO: 1-4 and has the same function as the sequence shown in SEQ ID NO: 1-4.

[0038] In some embodiments of the invention, the identity is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71% or 70%.

[0039] In the present invention, the phrase "having the same function" refers to having at least one of similar properties or functions such as immunogenicity, binding activity, specificity, or screening effect. In some embodiments of the present invention, the difference in the properties or functions is no more than 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0040] The second aspect of the present invention provides a biomaterial, wherein the biomaterial comprises at least one of the following (1)-(3):

[0041] (1) a nucleic acid molecule encoding the fusion protein described in the above aspects;

[0042] (2) an expression vector containing the nucleic acid molecule described in (1);

[0043] (3) A transformant containing the nucleic acid molecule described in (1) and / or the expression element described in (2).

[0044] In some embodiments of the present invention, the transformants do not involve animal or plant reproductive materials.

[0045] In some embodiments of the present invention, the expression vector includes viruses, plasmids and phages.

[0046] In the present invention, the term "expressor" refers to a vector or expression system used to integrate or insert a foreign gene of interest.

[0047] In some embodiments of the present invention, the expression subunit further includes at least one of: a promoter, an enhancer sequence, a replication origin, a terminator, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, and a reporter gene.

[0048] In some embodiments of the present invention, the virus includes lentivirus, adenovirus, retrovirus and adeno-associated virus.

[0049] In some embodiments of the present invention, the transformants include bacteria (such as Escherichia coli or Bacillus subtilis, etc.), fungi (such as yeast or Aspergillus, etc.), and animal and plant cells.

[0050] In some embodiments of the present invention, the transformant further comprises insect cells, such as S2 Drosophila cells or Sf9 cells.

[0051] In the present invention, the term "transformant" refers to a recipient cell that has acquired a new genetic marker after incorporation or introduction of a foreign gene.

[0052] In some embodiments of the present invention, the construction of the expression vector and the transformant can be achieved based on any conventional technical means in the art.

[0053] The third aspect of the present invention provides the use of the fusion protein or biomaterial described in the above aspects in preparing antigens of coronavirus and / or its mutants.

[0054] In some embodiments of the present invention, the antigen includes complete antigens and incomplete antigens (such as haptens, etc.).

[0055] The fourth aspect of the present invention provides the use of the fusion protein or biomaterial described in the above aspects in preparing antibodies against coronaviruses and / or their mutants.

[0056] In some embodiments of the present invention, the antibodies include, but are not limited to, at least one of: a monoclonal antibody (mAb), a polyclonal antibody, a chimeric antibody, a multispecific antibody, an antibody fragment (such as a Fab fragment, F(ab')2 and a single domain antibody (VHH)) and a conjugated antibody (such as an antibody-drug conjugate (ADC)).

[0057] In some embodiments of the present invention, the antibodies against coronavirus and / or its mutants are multispecific and can specifically bind to multiple coronaviruses and / or their mutants.

[0058] A fifth aspect of the present invention provides a method for preparing antibodies against coronavirus and / or its mutants, comprising:

[0059] The fusion protein or biological material described in the above aspects is used as an immunogen to immunize an animal, and the serum of the immunized animal is extracted to obtain the product.

[0060] In some embodiments of the invention, the animal is immunized multiple times.

[0061] In some embodiments of the present invention, the animals include but are not limited to: primates (including macaques, baboons, chimpanzees, marmosets, etc.), dogs, rabbits, sheep (such as goats and sheep), horses, and rodents (such as rats and mice).

[0062] The sixth aspect of the present invention provides the use of the fusion protein or biomaterial described in the above aspects in the preparation of a drug for treating and / or preventing coronavirus and / or its mutants.

[0063] In some embodiments of the invention, the medicament comprises a vaccine.

[0064] The beneficial effects of the present invention are:

[0065] 1. The present invention has explored and obtained multiple new glycosylation modification sites for improving the immunogenicity and broad spectrum of coronavirus RBD, and constructed a chimeric RBD-HRC trimer antigen based on them.

[0066] 2. The present invention provides a design strategy for a chimeric RBD-HRC trimer antigen, which forms a simplified version of the S protein by forming a trimer of RBD and HRC, thereby inducing a sustained humoral immune response and broad-spectrum neutralizing antibodies, significantly increasing the immunogenicity of the antigen, thereby triggering the body to produce more neutralizing antibodies and a stronger immune response. By further chimerizing the glycosylated RBDs of different coronaviruses and their variants, cross-immune reactions can be further induced, thereby activating stronger immune responses against multiple virus variants at the same time, improving the broad spectrum and long-term effectiveness of the vaccine. The present invention dually verified the feasibility of the chimeric RBD-HRC trimer antigen as a vaccine through antibody titer detection and pseudovirus neutralization effect experiments, and confirmed that it has immune activity against multiple coronaviruses such as SARS-CoV-2, SARS-CoV-2-Omicron, SARSCoV, MERS-CoV, SARS-CoV-2-D614G, SARS-CoV-2-JN.1, BANAL-52, BANAL-103 and HCoV-229E, and has excellent vaccine broad spectrum. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Schematic diagram of the construction process of trimer chimeric nanoparticles.

[0068] Figure 2 This is the map of the empty pLenti-6×HIS-puro plasmid in the examples.

[0069] Figure 3 Figure 3 shows the gel filtration chromatography curves of the nanoparticle proteins obtained after self-organization of the RBD-HRC-DN5B fusion proteins and I53-DN5A constructed in the examples. A represents the S2RHT-I53 nanoparticle protein; B represents the BA.5RHT-I53 nanoparticle protein; C represents the S2BA.5RHT-I53 nanoparticle protein; and D represents the S2SMRHT-I53 nanoparticle protein.

[0070] Figure 4 Transmission electron microscopy images (A), particle size characterization structures (B), and zeta potential (C) of the nanoparticle proteins obtained after self-organization of each RBD-HRC-DN5B fusion protein and I53-DN5A constructed in the examples.

[0071] Figure 5 This is a flow chart of the mouse immunization experiment of the nanoparticle protein in the embodiment of the present invention.

[0072] Figure 6 is the IgG antibody titer level after mice were immunized with the nanoparticle protein in the embodiment of the present invention; AD corresponds to S2WT-RBD-HRC-DN5B, SARS-RBD-HRC-DN5B, MERS-RBD-HRC-DN5B and BA.5-RBD-HRC-DN5B proteins as coating antigens, respectively.

[0073] Figure 7 These are the neutralizing antibody test results against different coronavirus pseudoviruses produced after mice were immunized with the nanoparticle protein in the embodiment of the present invention; wherein, AD corresponds to pseudoviruses of different coronaviruses, pseudoviruses of SARS-CoV-2 mutant strains, pseudoviruses of SARS-CoV-2-like coronaviruses, and pseudoviruses of alpha coronaviruses as objects, respectively.

[0074] Figure 8 These are the neutralizing antibody test results against the SARS-CoV-2 variant pseudovirus produced after mice were immunized with the nanoparticle protein in the embodiment of the present invention; wherein, AB correspond to SARS-CoV-D614G pseudovirus and SARS-CoV-2-JN.1 pseudovirus as objects, respectively.

[0075] Figure 9 These are the neutralizing antibody test results against SARS-CoV-2-like coronavirus pseudoviruses produced after mice were immunized with the nanoparticle protein in the embodiment of the present invention; wherein AB correspond to BANAL-52 pseudovirus and BANAL103 pseudovirus, respectively.

[0076] Figure 10 These are the results of neutralizing antibody testing against α-coronavirus pseudoviruses produced after mice were immunized with the nanoparticle protein in the examples of the present invention. DETAILED DESCRIPTION

[0077] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0078] Example 1

[0079] This embodiment provides a method for constructing trimer chimeric nanoparticles (schematic diagram as shown in FIG. Figure 1 As shown) and its characterization results, the specific steps are:

[0080] Chimera X software was used to compare the RBD structures of various novel coronaviruses (SARS-CoV-2-WT-RBD, SARS-CoV-RBD, MERS-CoV-RBD, Omicron-BA.5-RBD) with the native conformation of the S protein and perform immunogenic site analysis, screening and obtaining multiple special glycosylation sites.

[0081] Among them, the glycosylation modification sites of SARS-CoV-2-WT-RBD are R39N and P203N; the glycosylation modification sites of SARS-CoV-RBD are K28N and P191N; the glycosylation modification sites of MERS-CoV-RBD are V37N and T213N; and the glycosylation modification sites of Omicron-BA.5-RBD are R39N and P203N.

[0082] The conventional methods in the art are used to perform amino acid substitutions on the RBD of the above-mentioned new coronavirus according to the above-mentioned glycosylation modification sites to achieve glycosylation modification, thereby masking its non-neutralizing sites and exposing the neutralizing sites, thereby increasing the immunogenicity of the relevant RBD.

[0083] The RBD with optimized glycosylation modification was fused with the respective conserved domain HRC to form a simple RBD-HRC trimer structure.

[0084] The coding sequence of the RBD-HRC trimer and the coding sequence of I53-DN5B (the corresponding amino acid sequence of I53-DN5B is: MEEAELAYLLGELAYKLGEYRIAIRAYRIALKRDPNNAEAWYNLGNAYYKQGRYREAIEYYQKALELDP NNAEAWYNLGNAYYERGEYEEAIEYYRKALRLDPNNADAMQNLLNAKMREELE (SEQ ID NO: 5)) were inserted into the empty pLenti-6×HIS-puro plasmid (plasmid map shown in Figure 2The RBD-HRC trimer recombinant expression vectors (named as follows: pLenti-3F-SARS-CoV-2-WT-RBD-HRC-DN5B-6×HIS-puro, pLenti-3F-SARS-CoV-RBD-HRC-DN5B-6×HIS-puro, pLenti-3F-MERS-CoV-RBD-HRC-DN5B-6×HIS-puro, pLenti-3F-Omicron-BA.5-RBD-HRC-DN5B-6×HIS-puro) were obtained. The specific synthesis was handed over to Sangon Biotech (Shanghai) Co., Ltd. At the same time, RBD recombinant expression vectors expressing only unmodified SARS-CoV-2-WT and unmodified Omicron-BA.5 (named: pLenti-3F-SARS-CoV-2-WT-RBD-6×HIS-puro and pLenti-3F-Omicron-BA.5-RBD-6×HIS-puro, respectively) were constructed as subsequent experimental controls.

[0085] The obtained recombinant expression vector was packaged with lentivirus and then infected with 293F cells (human embryonic kidney cell-F clone) to construct a stable cell line. After the constructed stable cell line was cultured to 1L, the cell supernatant was collected by centrifugation at 4°C and 8000 rpm for 20 minutes, and phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1mM. The supernatant was then concentrated using 1×PBS in a concentration bucket and the buffer was exchanged. Impurities were removed by filtration through a 0.22μm filter membrane. The resulting concentrate was purified by nickel affinity column chromatography. The specific purification procedure was as follows: at 4°C, the concentrate was passed through a nickel affinity column (Histrap, GE Healthcare) and washed with buffer A (20mM Tris, 500mM NaCl, pH 7.4) to remove nonspecifically bound proteins. The target protein was then eluted with buffer B (20mM Tris, 500mM NaCl, pH 7.4, 500mM imidazole). The eluate was concentrated using a 10kDa concentrator and exchanged with buffer A (reduced by more than 30 times) to a final volume of less than 1mL. Finally, the target protein was further purified by molecular sieve chromatography using a Superdex™ 200 Increase 10 / 300GL column (GE Healthcare). The molecular sieve chromatography buffer used was 20mM Tris, 200mM NaCl, pH 7.4.

[0086] Recombinantly expressed, purified unmodified SARS-CoV-2-WT-RBD and unmodified Omicron-BA.5-RBD were obtained using the same procedures described above. The resulting monomeric SARS-CoV-2-WT-RBD (hereafter referred to as S2WT-RBD) and Omicron-BA.5-RBD (hereafter referred to as BA.5-RBD) RBDs will serve as control groups.

[0087] The amino acid sequence of the purified RBD-HRC-DN5B fusion protein is shown below, where the bold and underlined amino acid residues are glycosylation mutation sites.

[0088] SARS-CoV-2-WT-RBD-HRC-DN5B (hereinafter referred to as S2WT-RBD-HRC-DN5B):

[0089]

[0090] SARS-CoV-RBD-HRC-DN5B (hereinafter referred to as SARS-RBD-HRC-DN5B):

[0091]

[0092] MERS-CoV-RBD-HRC-DN5B (hereinafter referred to as MERS-RBD-HRC-DN5B):

[0093]

[0094] Omicron-BA.5-RBD-HRC-DN5B (hereinafter referred to as BA.5-RBD-HRC-DN5B):

[0095]

[0096] According to the above method, a DN5A recombinant expression plasmid (pET-32a-DN5A-6×HIS) was constructed based on the commercially available pET-32a vector and the I53-DN5A coding sequence (the amino acid sequence corresponding to I53-DN5A is: MGKYDGSKLRIGILHARWNAEIILALVLGALKRLQEFGVKRENIIIETVPGSFELPYGSKLFVEKQKRLGKPLDAIIPIGVLIKGSTMHFEYICDSTTHQLMKLNFELGIPVIFGVLTCLTDEQAEARAGLIEGKMHNHGEDWGAAAVEMATKFN (SEQ ID NO: 6)). The plasmid was then introduced into Escherichia coli BL21 competent cells. After plating, a single colony was picked and plated in 20 mL of LB liquid medium with ampicillin resistance and cultured in a bacterial shaker incubator at 37°C for 12-16 h. After the culture was completed, 20 mL of the bacterial solution was inoculated into 2 L of ampicillin-resistant LB liquid medium at a ratio of 1:100 and cultured in a bacterial shaker incubator at 37°C for 2.5 h. When the OD600 was 0.6-0.8, 1 mL of 1 M isopropyl-β-D-thiogalactopyranoside (IPTG) solution (final concentration 0.5 mM) was added and the cells were induced at low temperature for 14 h in a bacterial shaker at 16°C. The cells were centrifuged at 8000 rpm for 5 min at 4°C, the supernatant was removed, and the bacterial pellet was resuspended in protein purification buffer A (20 mM Tris, 500 mM NaCl pH 8.0). The bacteria were then disrupted using a low-temperature ultrahigh-pressure continuous flow cell disruptor (disrupted until the liquid was transparent and clear). The disrupted liquid was collected and centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was collected and filtered through a vacuum filtration device and a 0.22 μM filter membrane to complete sterilization and impurity removal, and then purified using nickel affinity column chromatography and molecular sieve chromatography to obtain purified I53-DN5A protein. The specific purification steps were the same as above.

[0097] The RBD-HRC-DN5B fusion protein and I53-DN5A protein purified in the above step were assembled at a 1:1 molar ratio in self-assembly buffer (50 mM HEPES, 300 mM NaCl, pH 8.0) using a rotary mixer at room temperature for 1 hour. After mixing, the mixture was centrifuged at 15,000 rpm for 5 minutes at 4°C. The supernatant was collected and passed through a gel filtration column coupled with a protein purification instrument to obtain nanoparticle protein.

[0098] in, Figure 3 The gel filtration chromatography curves of the nanoparticle proteins obtained after self-organization of each RBD-HRC-DN5B fusion protein and I53-DN5A are shown as examples. Specifically, Figure 3Figure A is the gel filtration chromatography curve of the nanoparticle protein (S2RHT-I53) obtained after self-organization of S2WT-RBD-HRC-DN5B and DN5A. Peak 1 is the successfully assembled S2RHT-I53 nanoparticle protein, Peak 2 is the excess SARS-CoV-2-WT-RBD-HRC-DN5B protein, and Peak 3 is the excess DN5A protein. Figure 3 Figure B shows the gel filtration chromatography curve of the nanoparticle protein (BA.5RHT-I53) obtained after self-assembly of Omicron-BA.5-RBD-HRC-DN5B and DN5A. Peak 1 represents the successfully assembled BA.5RHT-I53 nanoparticle protein, Peak 2 represents excess Omicron-BA.5-RBD-HRC-DN5B protein, and Peak 3 represents excess DN5A protein.

[0099] Figure 4 Figure A illustrates examples of negatively stained samples prepared using each of the prepared nanoparticle proteins and observed under a transmission electron microscope. For the negatively stained sample prepared using the S2RHT-I53 protein, it can be seen that the S2RHT-I53 protein nanoparticles prepared using the methods described in the above examples exhibit good uniformity, with a particle size of approximately 400 nm. For the negatively stained sample prepared using the BA.5RHT-I53 protein, it can be seen that the BA.5RHT-I53 protein nanoparticles prepared using the methods described in the above examples exhibit good uniformity, with a particle size of approximately 400 nm.

[0100] When two or three RBD-HRC-DN5B fusion proteins need to be chimeric, only the molar ratio of the RBD-HRC-DN5B fusion protein and DN5A needs to be adjusted. Specifically, when preparing the dual-chimeric S2BA.5RHT-I53 nanoparticle protein, the molar ratio of SARS-CoV-2-WT-RBD-HRC-DN5B, Omicron-BA.5-RBD-HRC-DN5B and DN5A is 1:1:2. The gel filtration chromatography curve is as follows Figure 3 As shown in Figure C, peak 1 is the successfully assembled S2BA.5RHT-I53 nanoparticle protein, peak 2 is the excess SARS-CoV-2-WT-RBD-HRC-DN5B and BA.5-RBD-HRC-FP-DN5B proteins, and peak 3 is the excess DN5A protein. The transmission electron microscopy image is shown in Figure 4. Figure 4As shown in Figure 2, the prepared S2BA.5RHT-I53 nanoparticle protein has good uniformity and a particle size of about 400 nm. When preparing the tri-chimeric S2SMRHT-I53 nanoparticle protein, the molar ratio of SARS-CoV-2-WT-RBD-HRC-DN5B, SARS-CoV-RBD-HRC-DN5B, MERS-CoV-RBD-HRC-DN5B and DN5A is 1:1:1:3. The gel filtration chromatography curve is shown in Figure 2. Figure 3 As shown in D in Figure 1, peak 1 is the successfully assembled S2SMRHT-I53 nanoparticle protein, peak 2 is the excess SARS-CoV-2-WT-RBD-HRC-DN5B, SARS-CoV-RBD-HRC-DN5B, MERS-CoV-RBD-HRC-DN5B protein, and peak 3 is the excess DN5A protein. Its transmission electron microscopy image is shown in Figure 1. Figure 4 As shown, the prepared S2SMRHT-I53 nanoparticle protein has good uniformity and a particle size of about 400 nm.

[0101] Thus, based on the method in this embodiment, chimeric protein nanoparticles with good uniformity and similar particle size can be obtained. Among them, although only S2RHT-I53 (based on a trimer of glycosylated SARS-CoV-2-WT-RBD), BA.5RHT-I53 (based on a trimer of glycosylated Omicron-BA.5-RBD), S2BA.5RHT-I53 (based on two trimers of glycosylated SARS-CoV-2-WT-RBD and glycosylated Omicron-BA.5-RBD), and S2SMRHT-I53 (based on three trimers of glycosylated SARS-CoV-2-WT-RBD, glycosylated SARS-CoV-RBD and glycosylated MERS-CoV-RBD) are exemplified, those skilled in the art can also prepare other nanoparticle proteins based on the self-organization of the above-mentioned glycosylated BRD-HRC-DN5B trimer and DN5A based on this method.

[0102] Example 2

[0103] In this example, the inventors used the nanoparticle protein obtained in the above example to immunize BALB / c mice (purchased from Weitonglihua Company) to verify the immunogenicity of the nanoparticle protein. The specific experimental steps are as follows:

[0104] Forty healthy female BALB / c mice aged 6-8 weeks were randomly divided into eight groups of five mice each. The mouse groups, immunogens, and vaccine doses are shown in Table 1. S2WT-RBD and BA.5-RBD proteins served as experimental controls, and PBS served as a negative control.

[0105] Table 1 Experimental animal groups and immunization status

[0106]

[0107] Mouse experimental procedures Figure 5 As shown, all immunizations were performed subcutaneously. All mice received a primary immunization on day 1 and a booster immunization on day 21, with a 200 μL injection volume for each immunization. At 2, 4, and 5 weeks after the primary immunization, the mice were tail-cuffed and blood was collected from the tail vein. The collected blood samples were placed in a 37°C incubator for 30 minutes and then refrigerated at 4°C for 4 hours. The upper serum layer was collected by centrifugation at 3000 rpm for 10 minutes at 4°C and stored at -80°C for determination of antigen-specific antibody titers and pseudovirus neutralization titers.

[0108] Antigen-specific antibody titers were detected using an enzyme-linked immunosorbent assay (ELISA). The specific testing method was as follows: the corresponding S2WT-RBD, BA.5-RBD, S2WT-RBD-HRC-DN5B, SARS-RBD-HRC-DN5B, MERS-RBD-HRC-DN5B, and BA.5-RBD-HRC-DN5B proteins were diluted to 2 μg / mL with ELISA coating buffer (0.05 M Na2CO3, 0.05 M NaHCO3, pH 9.6), then added to a 96-well ELISA plate at a volume of 100 μL / well and incubated at 4°C overnight. The coating buffer was discarded, and 200 μL of 1×PBST was added to each well to wash the plate. After standing for 1 minute, the plate was patted dry on a clean paper and washed four times. 200 μL of ELISA blocking buffer (2% BSA dissolved in PBST) was added and blocked at 37°C for 2 hours. Discard the blocking solution and repeat the plate washing method 4 times. 2 , 10 3 and 10 4 Perform serial dilutions at multiples of 100 μL / well and add to the ELISA plate. Incubate at 37°C for 1 hour. Discard the serum sample and repeat the plate washing method 4 times. Add 100 μL / well of ELISA antibody diluent (1% BSA dissolved in PBST) diluted 2×10 4 Incubate with 100 μL of HRP-conjugated Goat Anti-Mouse IgG at 37°C for 1 hour. Discard the solution and repeat the plate wash procedure four times. Add 100 μL / well of substrate colorimetric solution (Solarbio) and develop for 15 minutes at 37°C in the dark. Terminate the reaction by adding 50 μL of stop solution (Solarbio). Measure the OD450 value of each experimental group using a microplate reader.

[0109] The results are as follows Figure 6 shown.

[0110] Figure 6 The experimental results of RBD-specific antibody titers of serum after two immunizations. From the results, it can be seen that the serum of mice immunized with protein nanoparticles in each group can produce S2WT-RBD-HRC-DN5B, SARS-RBD-HRC-DN5B, MERS-RBD-HRC-DN5B and BA.5-RBD-HRC-DN5B-specific antibodies, and the antibody titers after the second immunization are more significant than those after the first immunization. However, no specific antibodies were produced in the control group (S2WT-RBD and BA.5-RBD) and the PBS group, indicating that the protein nanoparticles prepared by the above-mentioned embodiment method as a vaccine have more significant immunogenicity than pure RBD.

[0111] Example 3

[0112] In this example, the inventors used a pseudovirus neutralization experiment to further verify the effectiveness and broad-spectrum neutralization effect of protein nanoparticles as a vaccine.

[0113] The SARS-CoV, MERS-CoV, SARS-CoV-2-JN.1 and HCoV-229E pseudoviruses used in the examples were from Professor Wan Yushun's research group at Chongqing Medical University.

[0114] Other pseudoviruses were prepared in the inventor's laboratory. The specific preparation methods are as follows:

[0115] A biosynthesis company was commissioned to synthesize plasmids expressing SARS-CoV-2 S protein, Omicron S protein, BANAL-52S protein, and BANAL-103S protein, as well as a plasmid expressing the D614G mutation of the SARS-CoV-2 S protein (corresponding to the NCBI database numbers GeneID: 43740568, GeneID: 1489668, MZ937000.1, and MZ937001.1, respectively). HEK293T cells were transfected with the above-constructed viral S protein expression plasmids and the commercially available packaging plasmids pSPAX2 and pLenti-luc at a ratio of 1:1:1 using PEI for 6 hours. After transfection is completed, the culture medium in the well plate is discarded and replaced with fresh culture medium. After continuing to culture for 72 hours, the cell culture supernatant in the well plate is collected and centrifuged at 12000 rpm for 5 minutes. The supernatant is collected in a sterile 2 mL centrifuge tube to obtain SARS-CoV-2-WT, SARS-CoV-2-Omicron, BANAL-52, BANAL103 and SARS-CoV-2-D614G pseudoviruses.

[0116] The specific experimental method of the pseudovirus neutralization experiment is as follows:

[0117] The serum samples collected two weeks after the second immunization in the above example were inactivated in a 56°C water bath for 30 minutes. The inactivated serum was diluted with 1×PBS at a ratio of 1:100, and then the pseudoviruses of the above coronaviruses (SARS-CoV-2-WT, SARS-CoV-2-Omicron, SARS-CoV and MERS-CoV), pseudoviruses of SARS-CoV-2 mutants (SARS-CoV-2-D614G and SARS-CoV-2-JN.1), pseudoviruses of SARS-CoV-2-like coronaviruses (BANAL-52, BANAL103) and pseudoviruses of alpha coronaviruses (HCoV-229E) were mixed with the diluted serum at a volume ratio of 1:1 and placed in a cell culture incubator for 1 hour. Four replicate wells were set up for each experimental group, and essentially equal amounts of 293T-hACE2 cells were added and cultured for 48 hours. After the culture was completed, the culture medium was aspirated and discarded, 40 μL of Luciferase lysis solution was added to each well, and the cells were placed on a shaker at 150 rpm for 5 minutes. After thorough shaking, the lysates from the 96-well plate were transferred to a fresh 96-well plate using a pipette and 40 μL of substrate (Promega, Luciferase 1000 Assay System) was added. The results were read using a microplate reader and processed to analyze the broad-spectrum activity of the protein nanoparticle vaccine. A negative control (serum alone) and a positive control (pseudovirus alone) were included as reference controls.

[0118] The results are as follows Figure 7-10 shown.

[0119] Figure 7-10 These are the results of neutralizing antibody detection against pseudoviruses of the above-mentioned different coronaviruses, pseudoviruses of SARS-CoV-2 mutant strains, pseudoviruses of SARS-CoV-2-like coronaviruses, and pseudoviruses of alpha coronaviruses produced in the serum of mice in each experimental group two weeks after the second immunization.

[0120] like Figure 7As shown, the S2WT-RBD and BA.5-RBD groups showed some significance compared to the PBS group, but the three groups were still on the same order of magnitude. The sera of the S2RHT-I53, BA.5RHT-I53, and S2BA.5RHT-I53 groups contained specific neutralizing antibodies that bound to SARS-CoV-2-WT and SARS-CoV-2-Omicron pseudoviruses, preventing the corresponding viruses from invading cells. The level of neutralizing antibodies specifically binding to the SARS-CoV-2-WT pseudovirus in the serum of mice in the S2BA.5RHT-I53 group was approximately 5 times that of the chimeric single antigen group, and the level of neutralizing antibodies specifically binding to the SARS-CoV-2-Omicron pseudovirus was approximately 10 times that of the chimeric single antigen group. Moreover, surprisingly, although the S2BA.5RHT-I53 protein nanoparticles do not contain antigens of the two coronaviruses SARS-CoV and MERS-CoV, the results showed that the content of neutralizing antibodies that specifically bind to SARS-CoV and MERS-CoV pseudoviruses in the serum of mice immunized with S2BA.5RHT-I53 protein nanoparticles was higher than that of single chimeric protein nanoparticle antigens. This shows that the dual-chimeric protein nanoparticles in the above examples are more immunogenic than single chimeric antigens, and can stimulate the production of more specific neutralizing antibodies against SARS-CoV-2 and its mutants in mice, thereby enhancing the broad spectrum of the chimeric protein nanoparticle vaccine. In addition, it can be found that the content of neutralizing antibodies that specifically bind to SARS-CoV and MERS-CoV pseudoviruses in the serum of mice in the S2SMRHT-I53 group is the highest among all groups, while the content of neutralizing antibodies that specifically bind to SARS-CoV-2-WT and SARS-CoV-2-Omicron pseudoviruses is basically consistent with that of the single chimeric protein nanoparticle antigen, which also shows that the three-chimeric protein nanoparticles in the above example have stronger immunogenicity than the single chimeric protein nanoparticle antigen.

[0121] like Figure 8As shown, for the SARS-CoV-2-D614G pseudovirus, the S2WT-RBD and BA.5-RBD groups showed some significant differences compared to the PBS group, but the three groups were still on the same order of magnitude. For the SARS-CoV-2-JN.1 pseudovirus, neither the S2WT-RBD nor the BA.5-RBD groups showed specific neutralizing antibodies in their serum, compared to the PBS group, that could bind to the pseudovirus and prevent it from invading cells. In contrast, the serum of the protein nanoparticle group contained specific neutralizing antibodies that bound to both SARS-CoV-2-D614G and SARS-CoV-2-JN.1 pseudoviruses. The level of neutralizing antibodies specifically binding to the SARS-CoV-2-D614G pseudovirus in the serum of mice in the S2BA.5RHT-I53 group was approximately 100-fold higher than that of the single chimeric protein nanoparticle antigen, and approximately twice as high as that of the single chimeric protein nanoparticle antigen. The content of neutralizing antibodies that specifically bind to SARS-CoV-2-D614G pseudovirus in the serum of mice in the S2SMRHT-I53 group was much higher than that in the single chimeric protein nanoparticle antigen group, while the content of neutralizing antibodies that specifically bind to SARS-CoV-2-JN.1 pseudovirus was basically the same as that in the single chimeric protein nanoparticle antigen group.

[0122] like Figure 9 As shown, for the BANAL-52 pseudovirus, the S2WT-RBD and BA.5-RBD groups showed some significance compared to the PBS group, but the three groups were still on the same order of magnitude. For the BANAL-103 pseudovirus, the S2WT-RBD and BA.5-RBD groups showed some significance compared to the PBS group, especially the S2WT-RBD group, but the efficacy of both groups was still far lower than that of the various protein nanoparticle groups. The serum of each protein particle group contained specific neutralizing antibodies that bound to the BANAL-52 and BANAL103 pseudoviruses, preventing them from invading cells. The level of neutralizing antibodies specifically binding to the BANAL-52 and BANAL103 pseudoviruses in the serum of mice in the S2BA.5RHT-I53 group was higher than that in the single chimeric protein nanoparticle antigen group. The content of neutralizing antibodies bound to BANAL-52 and BANAL103 pseudoviruses in the serum of mice in the S2SMRHT-I53 group was basically the same as that in the single chimeric protein nanoparticle antigen group. The reason may be that the genomes of BANAL-52 and BANAL-103 are similar to those of SARS-CoV-2, while the amount of chimeric SARS-CoV-2 antigen in S2SMRHT-I53 is lower than that of S2RHT-I53 and BA.5RHT-I53.

[0123] like Figure 10As shown, the S2WT-RBD and BA.5-RBD groups showed some significant efficacy against the HCoV-229E pseudovirus compared to the PBS group, particularly the BA.5-RBD group, but the efficacy was still far lower than that of the various protein nanoparticle groups. The serum of mice in each chimeric protein nanoparticle group contained specific neutralizing antibodies that bound to the HCoV-229E pseudovirus, preventing it from invading cells. The serum of mice in the S2BA.5RHT-I53 group contained higher levels of neutralizing antibodies specifically binding to the HCoV-229E pseudovirus than those in the S2RHT-I53 and BA.5RHT-I53 groups. The serum of mice in the S2SMRHT-I53 group had the highest level of neutralizing antibodies against HCoV-229E among the various chimeric protein nanoparticle groups.

[0124] In summary, it can be found that the protein nanoparticle vaccine prepared according to the method in the embodiment of the present invention has high immunogenicity to different coronaviruses, SARS-CoV-2D614G variant, early Omicro variant subtype BA.5, and later Omicron variant subtype JN.1 variant, and can effectively stimulate the production of specific neutralizing antibodies against horseshoe bat coronavirus and alpha coronavirus in mice. Moreover, the triple-chimeric protein nanoparticle antigen has stronger immunogenicity than the single-chimeric and double-chimeric protein nanoparticle antigens, and also increases the broad spectrum of coronaviruses, indicating that it has a balanced and effective immune protection effect against multiple coronaviruses and variants, confirming that the scheme of constructing a broad-spectrum chimeric protein nanoparticle vaccine for coronavirus using the method in the embodiment of the present invention is effective and feasible, and has broad application prospects.

[0125] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein comprises: glycosylated modified coronavirus RBD sequence, coronavirus HRC and DN5B sequence; The novel coronavirus includes at least one of SARS-CoV-2, SARS, MERS wild type and their variants; Preferably, the fusion protein consists of a glycosylation-modified coronavirus RBD sequence, a coronavirus HRC, and a DN5B sequence connected in sequence.

2. The fusion protein according to claim 1, characterized in that The glycosylation modification includes at least one of the following: Glycosylation occurs at R39 and / or P203 of the wild-type SARS-CoV-2 RBD sequence; Glycosylation occurs at K28 and / or P191 positions of the SARS wild-type RBD sequence; Glycosylation occurs at positions V37 and / or T213 of the MERS wild-type RBD sequence; and Glycosylation modification occurs at the R39 and / or P203 positions of the RBD sequence of the SARS-CoV-2 variant Omicron-BA.

5.

3. The fusion protein according to claim 1, characterized in that The fusion protein contains glycosylation-modified RBD sequences of at least two coronaviruses.

4. The fusion protein according to claim 1, characterized in that The fusion protein further comprises a functional group; Preferably, the functional group comprises at least one of a linker, a reporter group, a protein tag, a carrier and a lipid group; Preferably, the protein tag comprises at least one of an affinity tag, an epitope tag and a fluorescent tag.

5. The fusion protein according to any one of claims 1 to 4, characterized in that The fusion protein comprises: (1) having a sequence as shown in any one of SEQ ID NOs: 1-4; (2) Sequences wherein one or more amino acids are substituted and / or deleted and / or added to SEQ ID NOs: 1-4 and have the same functions as the sequences shown in SEQ ID NOs: 1-4; (3) A sequence that is at least 90% identical to SEQ ID NO: 1-4 and has the same function as the sequence shown in SEQ ID NO: 1-4.

6. A biomaterial, characterized in that The biomaterial includes at least one of the following (1)-(3): (1) A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 5; (2) an expression vector containing the nucleic acid molecule described in (1); (3) A transformant containing the nucleic acid molecule described in (1) and / or the expression vector described in (2); wherein the transformants do not involve animal or plant reproductive materials; Preferably, the expression vector includes viruses, plasmids and phages; Preferably, the transformants include bacteria, fungi, animals and plants.

7. Use of the fusion protein according to any one of claims 1 to 5 or the biomaterial according to claim 6 in preparing antigens of coronaviruses and / or mutants thereof.

8. Use of the fusion protein according to any one of claims 1 to 5 or the biomaterial according to claim 6 in the preparation of antibodies against coronaviruses and / or mutants thereof; Preferably, the antibodies against coronavirus and / or its mutants are multispecific and can specifically bind to multiple coronaviruses and / or their mutants.

9. A method for preparing antibodies against coronavirus and / or its mutants, comprising: The fusion protein according to any one of claims 1 to 5 or the biological material according to claim 6 is used as an immunogen to immunize an animal, and the serum of the immunized animal is extracted to obtain the product.

10. Use of the fusion protein according to any one of claims 1 to 5 or the biomaterial according to claim 6 in the preparation of a drug for treating and / or preventing coronavirus and / or its mutants; Preferably, the medicament comprises a vaccine.

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