Novel coronavirus immunogenic compositions, methods of making and use thereof

Abstract

The novel coronavirus immunogenic composition of the present application comprises mixed receptor binding domains and N-terminal domains from different strain S proteins, can induce the production of neutralizing antibodies against different strains, experiments show that the novel coronavirus immunogenic composition of the present application has high immunogenicity, and can exhibit significantly improved immunization effect compared with monovalent antigen for different strains, and the bivalent or multivalent vaccine prepared by using the same is suitable as a candidate vaccine for coping with COVID-19, and has certain application potential for the current rapidly spreading Omicron mutant strain.

Description

Technical Field

[0001] This application belongs to the field of biomedical engineering technology, and specifically relates to a novel coronavirus immunogenic composition, its preparation method and application. Background Technology

[0002] Infection with the novel coronavirus (SARS-CoV-2) can lead to coronavirus disease (COVID-19). Common symptoms include fever, cough, and sore throat. In more severe cases, infection can cause difficulty breathing, hypoxemia, acute respiratory distress syndrome, and even death. The novel coronavirus can be transmitted from person to person through the respiratory tract and droplets, and there is also the possibility of transmission through the air and digestive tract.

[0003] The SARS-CoV-2 viral particle contains four structural proteins: the spike protein (S), nucleocapsid protein (N), membrane protein (M), and envelope protein (E). Studies have found that only antibodies targeting the S protein have neutralizing activity; therefore, all vaccines currently under development contain the S protein or its components. The receptor-binding domain of the S protein is considered the primary antigenic target region for inducing the production of neutralizing antibodies. Using the receptor-binding domain in vaccines allows the body to focus the generated neutralizing antibodies more precisely on the viral receptor, thus improving the immunogenicity and immunization efficiency of the vaccine. SARS-CoV-2 enters cells by binding to the host cell receptor hACE2 through its receptor-binding domain.

[0004] The novel coronavirus continues to evolve during its spread, and several representative mutant strains have been detected, such as the Alpha, Beta, Delta, and Omicron strains. Most currently developed or under-development novel coronaviruses can only target one strain and cannot simultaneously and effectively induce immunity against different strains. Summary of the Invention

[0005] The purpose of this invention is to provide a novel coronavirus immunogenic composition comprising a receptor-binding domain and an N-terminal domain of the S protein of at least two mixed strains. This novel coronavirus immunogenic composition has higher immunogenicity and can stimulate the production of neutralizing antibodies against different strains, thereby significantly improving the immune effect.

[0006] To achieve the objectives of this invention, in one aspect, this invention provides an immunogenic composition comprising a first component, the first component comprising at least one receptor-binding domain (RBD) of a novel coronavirus spike protein (S protein) or a functionally active fragment thereof; and a second component, the second component comprising at least one N-terminal domain (NTD) of a novel coronavirus spike protein or a functionally active fragment thereof. In this immunogenic composition, the RBD or its functionally active fragment and the NTD or its functionally active fragment are not fused, but are directly mixed.

[0007] In some embodiments, the novel coronavirus strain is selected from the following strains: the prototype strain, the Alpha strain, the Beta strain, the Gamma strain, the Delta strain, the Lambda strain, the Mu strain, and the Omicron strain.

[0008] The table below shows the correspondence between the World Health Organization and other naming systems for novel coronavirus strains.

[0009]

[0010] The newly discovered strain can also be found in [reference needed]. https: / / cov-lineages.org / lineage_list.html .

[0011] In some embodiments, the Omicron strain includes BA.1, BA.2, BA.3, BA.4, and BA.5 variants.

[0012] In some embodiments, the first component comprises 2 to 4 RBDs or functionally active fragments of the spike protein of novel coronavirus strains; the second component comprises 2 to 4 NTDs or functionally active fragments of the spike protein of novel coronavirus strains.

[0013] In some embodiments, each RBD or its functionally active fragment contained in the first component and each NTD or its functionally active fragment contained in the second component are derived from the same novel coronavirus strain, that is, they simultaneously have RBDs or their functionally active fragments and NTDs or their functionally active fragments from several strains.

[0014] For example, in some cases, the immunogenic composition contains an RBD or its functionally active fragment thereof, and an NTD or its functionally active fragment thereof derived from two novel coronavirus strains. In some embodiments, the first component contains the RBD of the prototype strain and the RBD of the Beta strain, and the second component contains the NTD of the prototype strain and the NTD of the Beta strain. In other embodiments, the first component contains the RBD of the prototype strain and the RBD of the Gamma strain, and the second component contains the NTD of the prototype strain and the NTD of the Gamma strain. In other embodiments, the first component contains the RBD of the prototype strain and the RBD of the Omicron strain, and the second component contains the NTD of the prototype strain and the NTD of the Omicron strain. In other embodiments, the first component contains the RBD of the Beta strain and the RBD of the Gamma strain, and the second component contains the NTD of the Beta strain and the NTD of the Gamma strain. In still other embodiments, the first component contains the RBD of the Delta strain and the RBD of the Omicron strain, and the second component contains the NTD of the Delta strain and the NTD of the Omicron strain.

[0015] In some cases, the immunogenic composition contains an RBD or its functionally active fragment thereof and an NTD or its functionally active fragment thereof derived from three novel coronaviruses. In some embodiments, the first component contains the RBD of the prototype strain, the RBD of the Delta strain, and the RBD of the Omicron strain, and the second component contains the NTD of the prototype strain, the NTD of the Delta strain, and the NTD of the Omicron strain. In other embodiments, the first component contains the RBD of the Alpha strain, the RBD of the Beta strain, and the RBD of the Delta strain, and the second component contains the NTD of the Alpha strain, the NTD of the Beta strain, and the NTD of the Delta strain. In still other embodiments, the first component contains the RBD of the Alpha strain, the RBD of the Lambda strain, and the RBD of the Omicron strain, and the second component contains the NTD of the Alpha strain, the NTD of the Lambda strain, and the NTD of the Omicron strain.

[0016] In some cases, the immunogenic composition contains an RBD or its functionally active fragment thereof and an NTD or its functionally active fragment thereof derived from four novel coronaviruses. In some embodiments, the first component contains the RBD of the prototype strain, the RBD of the Beta strain, the RBD of the Delta strain, and the RBD of the Mu strain, and the second component contains the NTD of the prototype strain, the NTD of the Beta strain, the NTD of the Delta strain, and the NTD of the Mu strain. In other embodiments, the first component contains the RBD of the Alpha strain, the RBD of the Beta strain, the RBD of the Delta strain, and the RBD of the Omicron strain, and the second component contains the NTD of the Alpha strain, the NTD of the Beta strain, the NTD of the Delta strain, and the NTD of the Omicron strain. In still other embodiments, the first component contains the RBD of the Beta strain, the RBD of the Gamma strain, the RBD of the Delta strain, and the RBD of the Omicron strain, and the second component contains the NTD of the Beta strain, the NTD of the Gamma strain, the NTD of the Delta strain, and the NTD of the Omicron strain.

[0017] In this invention, "first" and "second" are used only to indicate different types of antigens and do not indicate any order between the antigens.

[0018] In some embodiments, the amino acid sequence of the prototype strain RBD is SEQ ID NO:1, the amino acid sequence of the Beta strain RBD is SEQ ID NO:2, the amino acid sequence of the Gamma strain RBD is SEQ ID NO:3, the amino acid sequence of the Delta strain RBD is SEQ ID NO:4, the amino acid sequence of the Omicron BA.1 strain RBD is SEQ ID NO:5, the amino acid sequence of the BA.2 variant strain RBD is SEQ ID NO:6, the amino acid sequence of the BA.3 variant strain RBD is SEQ ID NO:7, and the amino acid sequences of the BA.4 and BA.5 variant strain RBDs are SEQ ID NO:8.

[0019] In some embodiments, the amino acid sequence of the prototype strain NTD is SEQ ID NO:9, the amino acid sequence of the Beta strain NTD is SEQ ID NO:10, the amino acid sequence of the Gamma strain NTD is SEQ ID NO:11, the amino acid sequence of the Delta strain NTD is SEQ ID NO:12, the amino acid sequence of the Omicron BA.1 strain NTD is SEQ ID NO:13, the amino acid sequence of the BA.2 variant strain NTD is SEQ ID NO:14, the amino acid sequence of the BA.3 variant strain NTD is SEQ ID NO:15, and the amino acid sequences of the BA.4 and BA.5 variant strain NTDs are SEQ ID NO:16.

[0020] In some embodiments, the RBD or its functionally active fragment in the first component is further linked to at least one of the following polypeptides: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, and hepatitis B surface antigen (HBsAg) or its functionally active fragment.

[0021] In some embodiments, the NTD or its functionally active fragment in the second component is further linked to at least one of the following polypeptides: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, and hepatitis B surface antigen (HBsAg) or its functionally active fragment.

[0022] In some embodiments, the polypeptide is fused within a frame to the RBD or a functional fragment thereof, and the polypeptide is fused within a frame to the NTD or a functional fragment thereof.

[0023] In some embodiments, the P2 or its functionally active fragment comprises an epitope peptide of tetanus toxin. For example, it may be expressed fused within the same open reading frame.

[0024] In some embodiments, the P2 or its functionally active fragment comprises an epitope peptide of tetanus toxin.

[0025] In some embodiments, the foldon domain or its functionally active fragment comprises amino acid residues at the C-terminus of phage T4 fibrin.

[0026] In some embodiments, the ferritin comprises Spodoptera litura ferritin or Helicobacter pylori ferritin.

[0027] In some embodiments, the P2 or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO:17-19; the foldon domain or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO:20-22; the *Spodoptera litura* ferritin comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO:23, and the light chain comprising the amino acid sequence shown in SEQ ID NO:24; the *Helicobacter pylori* ferritin or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO:25; and the hepatitis B surface antigen or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO:26.

[0028] In some implementations, the direct or indirect connection includes connection via connectors.

[0029] In some embodiments, the connector comprises a rigid joint, a flexible joint, or other sequences.

[0030] In some embodiments, the linker comprises an amino acid sequence shown in any one of SEQ ID NO:27-28.

[0031] In some embodiments, the weight ratio of the first component to the second component is (1-15):(1-15), preferably 1:1.

[0032] In some embodiments, the weight ratio of any two RBDs or their functionally active fragments in the first component is (1-5):(1-5), preferably 1:1.

[0033] In some embodiments, the weight ratio of any two NTDs or their functional active fragments in the second component is (1-5):(1-5), preferably 1:1.

[0034] Another aspect of the present invention provides a pharmaceutical composition comprising the above-described immunogenic composition and optionally a pharmaceutically acceptable excipient.

[0035] A third aspect of the present invention provides the use of the above-described immunogenic composition, or the above-described pharmaceutical composition, in the preparation of a vaccine.

[0036] In some implementations, the vaccine is used to prevent and / or treat COVID-19.

[0037] A fourth aspect of the present invention provides a method for preparing a COVID-19 subunit vaccine, comprising:

[0038] 1) Provide the above-mentioned immunogenic composition or pharmaceutical composition; and

[0039] 2) Mix the immunogenic composition or pharmaceutical composition described in 1) with a pharmaceutically acceptable adjuvant.

[0040] The fifth aspect of the present invention provides a COVID-19 subunit vaccine prepared according to the above method.

[0041] In some embodiments, each dose of vaccine contains 10-80 μg, preferably 20-50 μg, of the novel coronavirus immunogenic composition.

[0042] In some embodiments, the adjuvant is selected from one or more of aluminum adjuvants, MF59, MPL, QS-21, GLA, CpG, AS01, AS02, AS03, and AS04 adjuvants.

[0043] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0044] The novel coronavirus immunogenic composition of the present invention comprises a mixture of receptor-binding domains and N-terminal domains derived from the S protein of different strains, which can induce the production of neutralizing antibodies against different strains. Experiments show that the novel coronavirus immunogenic composition of the present invention has high immunogenicity and can exhibit significantly improved immune effects against different strains compared to monovalent antigens. The bivalent or multivalent vaccines prepared using it are suitable as a candidate vaccine for responding to COVID-19, and also have certain application potential against the currently rapidly spreading Omicron mutant strain. Attached Figure Description

[0045] Figure 1 The results shown are from a cell line passaging stability assay after transfection.

[0046] Figure 2 The image shows the results of SDS-PAGE electrophoresis and WB verification of the NTD-RBD-Foldon (prototype strain) protein. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Furthermore, unless otherwise specified, any instances where a specific strain is not mentioned in the embodiments are assumed to be wild-type strains (prototype strains), and any instances where a specific Omicron mutant strain is not mentioned are assumed to be BA.1 mutant strains.

[0048] Terminology definition:

[0049] In this application, the term "S protein," also known as "Spike protein" or "spike protein," generally refers to the capsid surface glycoprotein of coronaviruses. SARS-CoV-2 binds to the ACE2 receptor and invades cells via the S protein. The prototype S protein consists of 1273 amino acids and contains a transmembrane region comprising a segment from the N-terminus or from amino acid 14 of a coronavirus capsid surface glycoprotein up to amino acid 1273, or a corresponding region from other SAS viruses. The S1 protein is subunit 1 of the S protein, primarily referring to the segment from the N-terminus or amino acid 14 to amino acid 685.

[0050] In this application, the term "RBD" refers to the receptor-binding domain of the SARS-CoV-2 spike protein (S protein). For example, in this application, the RBD of the prototype strain may be a peptide of SARS-CoV-2 spike protein (S protein) between amino acids 310 and 560, or a mutant thereof, or may be truncated to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids from the N-terminus or C-terminus. In this application, the RBD of the prototype strain may be a peptide of SARS-CoV-2 spike protein (S protein) between amino acids 319 and 541, or a mutant thereof, or may be suitably truncated to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids from the N-terminus or C-terminus. In this application, the RBD of the prototype strain may be a peptide segment between amino acids 331 and 524 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof, or suitably truncated to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids at the N-terminus or C-terminus. In this application, "mutant" generally refers to a sequence that differs from the reference sequence due to the presence of one or more differences (mutations). This difference may be a substitution, deletion, or insertion of one or more amino acids. In some embodiments, the RBD may also be the RBD of a beta mutant, a gamma mutant, a delta mutant, or an omega mutant.

[0051] In this application, the term "NTD" refers to the N-terminal domain of the SARS-CoV-2 spike protein (S protein). For example, in this application, the NTD of the prototype strain can be a peptide of SARS-CoV-2 spike protein (S protein) between amino acids 13 and 353, or a mutant thereof, or may be suitably truncated to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids at the N-terminus or C-terminus. For example, the NTD of the prototype strain can be a peptide of SARS-CoV-2 spike protein (S protein) between amino acids 13 and 303, or a mutant thereof. In this application, the NTD of the prototype strain can be a peptide of SARS-CoV-2 spike protein (S protein) between amino acids 14 and 304, or a mutant thereof. In this application, the NTD of the prototype strain can be a peptide of SARS-CoV-2 spike protein (S protein) between amino acids 18 and 353, or a mutant thereof. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). These differences can be substitutions, deletions, or insertions of one or more amino acids. In some embodiments, the NTD can also be a beta mutant, a gamma mutant, or a delta mutant NTD.

[0052] In this application, the term "Foldon domain" generally refers to residues at the C-terminus of phage T4 fibrin. In this application, the Foldon domain can be the 27 residues at the C-terminus of phage T4 fibrin or a mutant. In this application, the Foldon domain can be a truncated or extended form obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of phage T4 fibrin. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). This difference can be a substitution, deletion, or insertion of one or more amino acids.

[0053] In this application, the term "P2" generally refers to an epitope peptide of tetanus toxin. For example, P2 can be a peptide of epitope peptides 830 to 844 of tetanus toxin or a mutant thereof. For example, P2 can be a peptide of epitope peptides 830 to 845 of tetanus toxin or a mutant thereof. For example, P2 can be a peptide of epitope peptides 829 to 844 of tetanus toxin or a mutant thereof. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids from the N-terminus or C-terminus of epitope peptides 829 to 844 of tetanus toxin. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids from the N-terminus or C-terminus of epitope peptides 830 to 845 of tetanus toxin. For example, P2 can be a peptide obtained by truncating or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of tetanus toxin epitope peptides 830 to 844. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). These differences can be substitutions, deletions, or insertions of one or more amino acids.

[0054] In this application, the terms "ferritin" generally refer to *Spodoptera litura* ferritin or *Helicobacter pylori* ferritin. In this application, *Spodoptera litura* ferritin has a heavy chain and a light chain (ferritin LC and ferritin HC). In this application, *Helicobacter pylori* ferritin has a single-chain structure. In this application, ferritin can be a mutant of *Spodoptera litura* or *Helicobacter pylori* ferritin. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). These differences can be substitutions, deletions, or insertions of one or more amino acids. In this application, "light chain of *Spodoptera litura* ferritin" and "LC" are used interchangeably with "ferritin LC". In this application, "heavy chain of *Spodoptera litura* ferritin" and "HC" are used interchangeably with "ferritin HC".

[0055] In this application, the term "hepatitis B surface antigen (HBsAg)" generally refers to a coat protein in the outermost envelope of the hepatitis B virus. For example, the amino acid sequence of the hepatitis B surface antigen can be the sequence corresponding to protein sequence accession numbers AAA45524, ANJ76941, CAA24234, or AAC34729 in NCBI, or it can be suitably truncated or augmented with 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus, or it can have protein mutations, such as deletions, substitutions, or insertions of one or more amino acids.

[0056] In this application, the term "functionally active fragment" generally refers to a fragment that has similar biological activity to RBD, NTD, Foldon domain, Fc domain, P2, ferritin, and hepatitis B surface antigen HBsAg.

[0057] In this application, the term "fusion protein" generally refers to a biologically functional protein molecule obtained through genetic engineering techniques. In this application, the fusion protein can be a fusion protein composed of an NTD or its functionally active fragment and an RBD or its functionally active fragment. In this application, the fusion protein can also be a fusion protein composed of an NTD or its functionally active fragment, an RBD or its functionally active fragment, and a Foldon domain, P2, ferritin, hepatitis B surface antigen HBsAg, or the Fc domain of human immunoglobulin. The Foldon domain, P2, ferritin, hepatitis B surface antigen HBsAg, or the Fc domain are directly linked to the RBD or its functionally active fragment or linked through a linker. The linker sequence can be GGSSG, etc.

[0058] In this application, the term "immunogenic composition" generally refers to a subunit composition. In this application, a subunit composition is a composition in which the components have been isolated and purified to a purity of at least 50%, 60%, 70%, 80%, or 90% before being mixed to form an antigenic composition. For example, the subunit composition may be an aqueous solution of a water-soluble protein. For example, the subunit composition may contain a detergent. For example, the subunit composition may contain non-ionic, zwitterionic, or ionic detergents. For example, the subunit composition may contain lipids.

[0059] In this application, the term "adjuvant" refers to a substance capable of enhancing or altering the type of immune response. The adjuvant in this application may be selected from one or more of aluminum adjuvant, MF59, MPL, QS-21, GLA, CpG, AS01, AS02, AS03, and AS04 adjuvant.

[0060] In this application, the term "weight ratio" generally refers to the weight ratio of the components of the immunogenic composition. In this application, each dose of the immunogenic composition may contain: 5-60 μg of a first component and 5-60 μg of a second component. For example, the immunogenic composition may contain 5 μg, 10 μg, 20 μg, 30 μg, or 40 μg of the first component. For example, the immunogenic composition may contain 5 μg, 10 μg, 20 μg, 30 μg, or 40 μg of the second component. In this application, the immunogenic composition may also contain three or more components.

[0061] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0062] In this application, the term "around" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0063] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers inserted nucleic acid molecules into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the functions described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0064] In this application, the term "host cell" generally refers to an individual cell, cell line, or cell culture that may contain or already contains a plasmid or vector including the nucleic acid molecules described in this application, or that is capable of expressing the antibodies or antigen-binding fragments described in this application. The host cell may include progeny of a single host cell. Due to natural, accidental, or intentional mutations, progeny cells may not necessarily be morphologically or genomically identical to the original parent cell, but they need to be capable of expressing the fusion protein described in this application. The host cell can be obtained by in vitro transfection of cells using the vector described in this application. The host cell can be a prokaryotic cell (e.g., *Escherichia coli*) or a eukaryotic cell (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells). In some embodiments, the host cell is a mammalian cell. For example, the mammalian cell may be a CHO cell.

[0065] Example 1: Construction of RBD-P2-6*HIS(GP101150-7) recombinant expression plasmid

[0066] Signal peptide addition: MGVPAVPEASSPRWGTLLLAIFLAASRGLVAA (SEQ ID NO: 56). Vector selection: pcdna3.1(+), codon optimized according to host CHO cells.

[0067] Cloning of the fragment. The primers for RBD-P2-6*HIS were used to amplify the fragment PCR product via PCR. This product was then recombined into the target vector pcdna3.1(+)(BamHI-XhoI digested vector) using a multi-segment recombination method, yielding the recombinant expression plasmid RBD-P2-6*HIS(GP101150-7). The reaction system is shown in Table 1.

[0068] Table 1. Reaction system for the ligation of the treated target fragment and the carrier.

[0069] Enzyme digestion vector 5μl Purified PCR products 5μl Seamless assembly MIX 10μl Total volume 20μl

[0070] The above ligation solution was ligated at a constant temperature of 52℃ for 30 min to obtain the recombinant expression plasmid RBD-P2-6*HIS(GP101150-7).

[0071] Transformation method: (1) Add 1-3 μl of the RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid at a concentration of 100 ng / μl to 100 μl of competent cells, gently shake and rotate to mix, and place on ice for 3 minutes. (2) Incubate in a 42℃ water bath for 90 seconds without shaking. (3) Place in an ice bath for about 3 minutes. (4) Add 500-800 μl of LB medium pre-warmed at 37℃ to each tube, and gently shake at 200 rpm on a 37℃ shaker for 40 minutes.

[0072] Verification of the recombinant expression plasmid RBD-P2-6*HIS(GP101150-7): (1) Prepare agar plates containing the corresponding antibiotics. (2) Take 100 μl of bacterial culture and spread it evenly on the agar plate containing the corresponding antibiotics. Use a sterile glass spreader to gently spread the bacteria on the surface of the plate and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours until colonies appear. (4) Pick bacteria from the plate, shake the plate at 250 rpm at 37°C for 14 hours, perform PCR identification using the bacterial culture, and send the positive clones for sequencing.

[0073] Identification method of cloned plasmid: PCR amplification of the RBD-P2-6*HIS fragment. The primer sequences were synthesized by the company's in-house primer department. The expected fragment length was 861 bp. The PCR reaction used a 20 μL system: 0.5 μL primer, 2 μL template bacterial culture, 0.5 μL polymerase buffer, 3 μL buffer, and 14 μL ddH2O. Cycling parameters: 96℃ pre-denaturation for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 20 s, 23 cycles, with a final extension at 72℃ for 1 min. Positive clones were screened by bacterial culture PCR. The obtained positive bacterial cultures were shaken at 37℃ to extract plasmids, which were then sequenced. Plasmids that matched the sequencing data were double-digested with BamHI-XhoI to obtain two fragments, 861 bp and 5372 bp.

[0074] RBD-P2-6*HIS(GP101150-7) recombinant expression plasmid extraction: *E. coli* cells (stbl3) containing 1% of the RBD-P2-6*HIS(GP101150-7) recombinant expression plasmid were inoculated into 2 ml LB medium and cultured overnight at 37°C with shaking. After cell treatment, 100 μg of plasmid was extracted using a plasmid extraction kit. The correctly aligned plasmid was sequenced and double-digested with BamHI-XhoI to obtain two fragments of 861 bp and 5372 bp.

[0075] Example 2: Cell transfection and purification of RBD-P2-6*HIS protein

[0076] During all cell manipulations, gently rotate the host cells to mix, avoiding vigorous mixing / pipetting. Subculture and expand CHO cells (from Thermo's EXPICHO) until a cell density of 4 × 10⁶ cells / year is reached. 6 –6×10 6 Cells / mL. Day -1: Cell expansion, expanding cultured cells to 3 × 10⁻⁶. 6 –4×10 6 Cells / mL, and allowed to grow overnight. Day 0: Transfect cells, determine viable cell density and survival rate; cell density should reach 7 × 10⁶ cells / mL. 6 –10×10 6 Transfect cells at a density of 95–99% viability using fresh expression medium preheated to 37°C, and dilute to a final density of 6 × 10⁶ cells / mL. 6Cells were cultured at 37°C with an incubator of 90 rpm at 50 mm amplitude and 8% CO2. Transfection reagent and the RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid complex were prepared using OPti-PRO SFM medium (4°C). For example, for 1 ml of CHO cells, 40 μl of OPti-PRO SFM was added, followed by 0.8 μg of RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid, and the mixture was incubated for 5 min. 40 μl of OPti-PRO SFM was then added, followed by 3 μl of Expi Fectamine CHO reagent, and the mixture was incubated for 5 min at room temperature for 1–5 minutes. The solution was then slowly transferred to a shaker flask with gentle shaking during the addition process. Cells were then incubated at 37°C with an incubator of 90 rpm at 50 mm amplitude and 8% CO2. 18–22 hours post-transfection, Enhancer and ExpiCHO feed were added, and standard experimental protocols were followed. Example: Add 6 μl of Enhancer and 0.24 ml of ExpiCHO Feed to 1 ml of cells, and incubate the cells at 37°C with a 50 mm amplitude incubator at 90 rpm and 8% CO2. Collect the cells 8 days after transfection for further purification.

[0077] Protein purification. The culture medium was centrifuged, and the supernatant was added to a Ni column. The column was incubated with shaking for 2 hours, followed by affinity chromatography purification using a gravity-fed empty column. Equilibration buffer: PBS, pH 7.4, 10 CV washes; Washing buffer: PBS, pH 7.4 with 20 mM Mimidazole, 10 CV washes; Elution buffer: PBS, pH 7.4 with 500 mM Mimidazole, 1 CV elution. This was repeated 5 times to obtain the purified RBD-P2-6*HIS protein, as shown in Table 2.

[0078] Table 2. Concentration and volume of purified protein

[0079] RBD-P2-6*HIS 0.05 1

[0080] SDS-PAGE electrophoresis and Western blotting verification. A. 1.0 mm PAGE gel, 8 μl sample loading, gel running sequence: M. Molecular weight marker. M. Culture medium. FT. Flow through. W. Wash. E. Eluted fractions. B. Electrophoresis: Mix RBD-P2-6*HIS purified protein with loading buffer (RBD-P2-6*HIS purified protein: 5 × loading buffer = 4:1), boil for 5 min, and then load the sample. First, perform electrophoresis at a constant voltage of 100V. You can see the marker gradually become a thin line. After the marker enters the separating gel, adjust the voltage to 300V and continue electrophoresis until the blue bromophenol blue band reaches the bottom of the gel (about 25 min). C. Transfer membrane: Pry open the glass plate and carefully remove the gel. Activate the PVDF membrane with methanol for 30 s, cut it and the filter paper to the same size as the gel block, and soak them in transfer buffer. From bottom to top: filter paper - PVDF membrane - gel - filter paper. Ensure there are no air bubbles between the layers, especially between the gel and the membrane. Pour all the transfer buffer from the petri dish into the transfer container. Transfer at a constant voltage of 20V; 20 minutes is required for a 50kDa protein. Larger molecular weight proteins require longer transfer times. D. Antibody incubation: After transfer, remove the PVDF membrane; you will see the marker transferred onto the membrane. Block with 5% skim milk (1g skim milk powder to 100ml PBST) for 1 hour, then wash with PBST 2×5min. Dilute the primary antibody (anti-His Mab) with 5% skim milk powder (1g skim milk powder, 100ml TBS), incubate in a small container, and react at 4℃ on a decolorizing shaker for 2 hours. Remove the PVDF membrane and wash with PBST 4×10min. Dilute the secondary antibody (goat anti-mouse) with 5% skim milk powder and incubate for 1 hour. Wash with PBST 4×10min. Use a shaker for all reactions. E. Color development, ECL color development: For each PBST membrane, mix 1 mL of solution A and 10 μL of solution B, drop the mixture onto the membrane, and record the image using a chemiluminescence imaging system after 5 minutes. Figure 1 As shown.

[0081] Example 3: Construction of NTD-P2-6*HIS(GP101150-8) recombinant expression plasmid

[0082] Signal peptide addition: MFVFLVLLPLVS (SEQ ID NO: 57), vector selection: pcdna3.1(+). Codon optimization was performed according to the host CHO cells. The NTD-P2-6*HIS primers were amplified by PCR to obtain the fragment PCR product, which was then recombined into the target vector pcdna3.1(+) (BamHI-XhoI digested vector) using a multi-segment recombination method to obtain the NTD-P2-6*HIS(GP101150-8) recombinant expression plasmid. The reaction system is shown in Table 3.

[0083] Table 3. Reaction system for the treatment of target fragment and carrier.

[0084] Enzyme digestion vector 5μl Purified PCR products 5μl Seamless assembly MIX 10μl Total volume 20μl

[0085] Transformation method: Add 1-3 μl of the NTD-P2-6*HIS(GP101150-8) recombinant expression plasmid at a concentration of approximately 100 ng / μl to approximately 100 μl of competent cells, gently shake and rotate to mix, and incubate on ice for 3 minutes. Incubate at 42°C for 90 seconds without shaking; incubate on ice for approximately 3 minutes; add 500-800 μl of pre-warmed LB medium at 37°C to each tube, and gently shake at 200 rpm on a 37°C shaker for 40 minutes.

[0086] Verification of the NTD-P2-6*HIS(GP101150-8) recombinant expression plasmid: (1) Prepare agar plates containing the corresponding antibiotics. (2) Take 100 μl of bacterial suspension and spread it evenly on the agar plate containing the corresponding antibiotics. Use a sterile glass spreader to gently spread the bacteria on the surface of the plate and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours until colonies appear. (4) Pick bacteria from the plate, shake the plate at 250 rpm at 37°C for 14 hours, perform PCR identification using the bacterial suspension, and send the positive clones for sequencing.

[0087] Identification of cloned plasmids: The NTD-P2-6*HIS fragment was amplified by PCR. The primer sequences were synthesized in-house by the company's primer department. The expected fragment length was 1005 bp. The PCR reaction used a 20 μL system: 0.5 μL primers, 2 μL template bacterial culture, 0.5 μL polymerase buffer, 3 μL buffer, and 14 μL ddH2O. Cycling parameters: 96℃ pre-denaturation for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 20 s, 23 cycles, with a final extension at 72℃ for 1 min. Positive clones were screened using bacterial culture PCR. Plasmids were extracted from the obtained positive bacterial cultures by shaking at 37℃ and sequenced. Plasmids with correct sequencing alignment were double-digested with BamHI-XhoI to obtain two fragments of 1005 bp and 5372 bp.

[0088] Extraction of the NTD-P2-6*HIS(GP101150-8) recombinant expression plasmid. *E. coli* cells (stbl3) containing 1% of the NTD-P2-6*HIS(GP101150-8) recombinant expression plasmid were inoculated into 2 ml of LB medium and cultured overnight at 37°C with shaking. After cell treatment, 100 μg of plasmid was extracted using a plasmid extraction kit. The correctly aligned plasmid was sequenced and double-digested with BamHI-XhoI to obtain two fragments of 1005 bp and 5372 bp.

[0089] Example 4 Cell transfection and purification of NTD-P2-6*HIS (GP101150-8) protein

[0090] During all cell manipulations, gently rotate to mix the host cells; avoid vigorous mixing / pipetting. Subculture and expand CHO cells until a cell density of 4 × 10⁶ cells / year is reached. 6 –6×10 6 Cells / mL. Day -1: CHO cell expansion, expanding cultured cells to 3 × 10⁻⁶. 6 –4×10 6 Cells / mL, and allowed to grow overnight. Day 0: Transfect cells, determine viable cell density and survival rate; cell density should reach 7 × 10⁶ cells / mL. 6 –10×10 6 Transfect cells at a density of 95–99% viability using fresh expression medium preheated to 37°C, and dilute to a final density of 6 × 10⁶ cells / mL. 6 Cells were cultured at 37°C with an incubator of 90 rpm at 50 mm amplitude and 8% CO2. Transfection reagent and NTD-P2-6*HIS (GP101150-8) recombinant expression plasmid complex were prepared using OPti-PRO SFM medium (4°C). For example, for 1 ml of cells, 40 μl of OPti-PRO SFM was added with 0.8 μg of NTD-P2-6*HIS (GP101150-8) recombinant expression plasmid, mixed, and incubated for 5 min; 40 μl of OPti-PRO SFM was added with 3 μl of ExpiFectamine CHO reagent, mixed, and incubated for 5 min at room temperature for 1–5 minutes. Then, the solution was slowly transferred to a shaker flask with gentle shaking during the addition process. Cells were then cultured at 37°C with an incubator of 90 rpm at 50 mm amplitude and 8% CO2. 18–22 hours after transfection, Enhancer and ExpiCHO feed were added, and standard experimental protocols were followed. Example: Add 6 μl Enhancer and 0.24 ml ExpiCHO Feed to 1 ml of cells, and incubate the cells at 37°C with a 50 mm amplitude incubator at 90 rpm and 8% CO2. Collect the cells 8 days after transfection for further purification.

[0091] Protein purification was performed by centrifugation of the culture medium, adding the supernatant to a Ni column, incubating with shaking for 2 hours, and then performing affinity chromatography using a gravity-fed empty column. Equilibration buffer: PBS, pH 7.4, 10 CV washes; Washing buffer: PBS, pH 7.4 with 20 mM Mimidazole, 10 CV washes; Elution buffer: PBS, pH 7.4 with 500 mM Mimidazole, 1 CV elution. This was repeated 5 times to obtain NTD-P2-6*HIS (GP101150-8) purified protein. The results are shown in Table 4.

[0092] Table 4. Concentration and volume of purified protein from NTD-P2-6*HIS (GP101150-8)

[0093] NTD-P2-6*HIS 0.03 1

[0094] SDS-PAGE electrophoresis and Western blotting verification. A. 1.0 mm PAGE gel, 8 μl sample volume, gel running sequence: M. Molecular weight marker. M. Culture medium. FT. Flow through. W. Wash. E. Eluted fractions. B. Electrophoresis: Mix NTD-P2-6*HIS (GP101150-8) purified protein with loading buffer (NTD-P2-6*HIS (GP101150-8) purified protein: 5 × loading buffer = 4:1), boil for 5 min, and then load the sample. First, perform electrophoresis at a constant voltage of 100 V. You can see the marker gradually become a thin line. After the marker enters the separating gel, adjust the voltage to 300 V and continue electrophoresis until the blue bromophenol blue band reaches the bottom of the gel (about 25 min). C. Transfer. Pry open the glass plate and carefully remove the gel. After activating the PVDF membrane with methanol for 30 seconds, cut it and the filter paper to the same size as the gel block, and soak it in transfer buffer. The order from bottom to top is: filter paper - PVDF membrane - gel - filter paper. Ensure there are no air bubbles between the layers, especially between the gel and the membrane. Pour all the transfer buffer from the petri dish into the transfer container. Transfer at a constant voltage of 20V; 50kDa protein requires 20 minutes. Larger molecular weight proteins require longer transfer times. D. Antibody incubation. After transfer, remove the PVDF membrane; the marker will be visible transferred to the membrane. Block with 5% skim milk (1g skim milk powder to 100ml PBST) for 1 hour, then wash with PBST 2×5min. Dilute the primary antibody (anti-His Mab) with 5% skim milk powder (1g skim milk powder to 100ml TBS), incubate in a small container, and react at 4℃ on a decolorizing shaker for 2 hours. Remove the PVDF membrane and wash with PBST 4×10min. The secondary antibody (goat anti-mouse) was also diluted with 5% skim milk powder and incubated for 1 hour. Washed with PBST 4 × 10 min. All measurements were taken using a shaker. E. Color development. ECL color development: For each PBST membrane, a mixture of 1 mL of solution A and 10 μL of solution B was dropped onto the membrane. After 5 min, the image was captured using a chemiluminescence imaging system. Figure 2 As shown.

[0095] Example 5: Construction of vector and protein expression

[0096] Referring to the above embodiments, the target fragment to be expressed was constructed as follows, and the protein was expressed and purified, as shown in Table 5.

[0097] Table 5 Different target fragments

[0098]

[0099]

[0100] Example 6 Preparation of the pharmaceutical composition

[0101] 0.04 mg / mL of RBD protein, 0.04 mg / mL of NTD protein; 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; and 5 mM to 25 mM histidine buffer at pH 5.0 to 7.0 were aliquoted into 2 ml vials, 0.5 ml per vial, and lyophilized.

[0102] Example 7 Preparation of the pharmaceutical composition

[0103] 0.04 mg / mL of RBD-P2 protein, 0.04 mg / mL of NTD-P2 protein; 2% to 15% (w / v) sorbitol; 0.01% to 0.05% (w / v) polysorbate 20; and 5 mM to 20 mM histidine buffer at pH 5.5 to 7.0 were aliquoted into 2 ml vials, 0.5 ml per vial, and lyophilized.

[0104] Example 8 Preparation of the pharmaceutical composition

[0105] 0.04 mg / mL of RBD protein, 0.04 mg / mL of NTD-P2 protein; 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; and 5 mM to 25 mM succinate buffer at pH 4.5 to 5.5 were aliquoted into 2 ml control bottles, 0.5 ml per bottle, and lyophilized.

[0106] Example 9 Preparation of the pharmaceutical composition

[0107] 0.04 mg / mL of RBD-P2 protein, 0.04 mg / mL of NTD protein; 2% to 15% (w / v) sorbitol; 0.01% to 0.05% (w / v) polysorbate 20; and 5 mM to 20 mM histidine buffer at pH 5.5 to 7.0 were aliquoted into 2 ml vials, 0.5 ml per vial, and lyophilized.

[0108] Example 10: Vaccine Preparation

[0109] The pharmaceutical composition of any one of Examples 6-9, wherein the composition further comprises aluminum hydroxide adjuvant at a concentration of 1 mg / mL.

[0110] The pharmaceutical composition described in any one of Examples 6-9 is supplemented with CpG1018 adjuvant (CpG was synthesized by Shanghai Sangon Biotech Co., Ltd. according to the CpG1018 sequence) at a concentration of 6 mg / mL.

[0111] Example 11 Vaccine Preparation

[0112] The pharmaceutical composition of any one of Examples 6-9 further comprises an adjuvant, for example, in an adjuvant vial of 0.5 ml, 50 μg of MPL, 500 μg of aluminum hydroxide, 150 mM of NaCl, 8 mM of disodium hydrogen phosphate dihydrate, and water for injection to a final volume of 0.5 ml.

[0113] Example 12 Vaccine Preparation

[0114] The pharmaceutical composition of any one of Examples 6-9, further comprising an adjuvant, for example, in a 0.25 ml adjuvant vial containing 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of Tween 80, 3.53 mg of NaCl, 0.09 mg of KCl, 0.51 mg of Na₂HPO₄, 0.09 mg of KH₂PO₄, and water for injection.

[0115] Example 13 Vaccine Preparation

[0116] The pharmaceutical composition of any one of Examples 6-9 further comprises an adjuvant, for example, an adjuvant vial of 0.5 ml containing 50 μg of MPL, 50 μg of QS-21, 1 mg of DOPC dioleoylphosphatidylcholine, 0.25 mg of cholesterol, 0.15 mg of anhydrous disodium phosphate, 0.54 mg of potassium dihydrogen phosphate, 4.385 mg of sodium chloride, and water for injection.

[0117] Example 14: Vaccine Preparation

[0118] The pharmaceutical composition of any one of Examples 6-9, further comprising an adjuvant, for example, in a 0.5 ml adjuvant vial containing 9.75 mg squalene, 1.175 mg Span 85, 1.175 mg Tween 80, 0.66 mg trisodium citrate dihydrate and 0.04 mg citrate monohydrate, and water for injection.

[0119] Example 15: Mouse Immunization Experiment with Different Antigens and Adjuvants

[0120] The antigen protein was combined with different adjuvants to immunize mice, and the level of protein-induced neutralizing antibodies was determined by ELISA. Mouse grouping and immunization regimens are shown in Table 6.

[0121] Table 6. Mouse grouping table

[0122]

[0123]

[0124] The NTD protein sequence is SEQ ID NO: 9, the RBD sequence is SEQ ID NO: 1, the Al(OH)3 adjuvant, CpG adjuvant, and CpG+Al(OH)3 adjuvant concentrations are as described in Example 10, the composition of AS01 is as described in Example 13, and the composition of AS04 is as described in Example 11. The results of mouse immunization 28 days later are shown in Table 7. The results show that RBD+NTD induces a stronger level of neutralizing antibodies.

[0125] Table 7 Results of mouse immunization 28 days later

[0126]

[0127] Example 16 Immunogenicity Evaluation Experiment of Different Antigens and Adjuvants

[0128] The antigen protein and MF59 adjuvant were used to immunize Japanese white rabbits. The level of neutralizing antibodies induced by the protein was determined by ELISA. Rabbit grouping and immunization regimen are shown in Table 8.

[0129] Table 8 Rabbit Grouping Table

[0130]

[0131]

[0132] The sequence of NTD-P2 is shown in SEQ ID NO: 48, the sequence of RBD-P2 is shown in SEQ ID NO: 49, the sequence of NTD-foldon is shown in SEQ ID NO: 50, the sequence of RBD-foldon is shown in SEQ ID NO: 51, the sequence of NTD-ferritin LC is shown in SEQ ID NO: 52, the sequence of RBD-ferritin is shown in SEQ ID NO: 53, the sequence of NTD-HBsAg is shown in SEQ ID NO: 54, the sequence of RBD-HBsAg is shown in SEQ ID NO: 55, and the adjuvant MF59 is shown in Example 14.

[0133] The test results 28 days after immunization are shown in Table 9. All groups were able to produce a certain level of neutralizing antibodies. The NTD-P2+RBD-P2 group was the best, significantly higher than the other groups. The NTD-ferritin+RBD-ferritin / NTD-HBsAg+RBD-HBsAg groups were the next best, indicating that the above fusion protein has a certain immunogenicity against the novel coronavirus.

[0134] Table 9. Test results of rabbits 28 days after immunization.

[0135]

[0136] Example 17 Immunogenicity Evaluation Experiment of Different Antigens and Adjuvants

[0137] The antigen protein was used in combination with different adjuvants to immunize rhesus monkeys, and the level of protein-induced neutralizing antibodies was determined by ELISA. The monkey groups and immunization regimens are as follows, with an inactivated COVID-19 vaccine (purchased from Beijing Institute of Biological Products Co., Ltd.) as the positive control. The test results of the monkeys 28 days after immunization are shown in Table 10.

[0138] Table 10. Immunization results in monkeys

[0139]

[0140]

[0141] The results showed that when AS03 adjuvant was used (refer to Example 12), the geometric mean titer was the highest and significantly higher than that of the other groups, indicating that AS03 adjuvant is a suitable adjuvant for the novel coronavirus vaccine provided by the present invention.

[0142] Example 18: Detection of the effect of recombinant COVID-19 vaccine candidate antigen on inducing neutralizing antibody production in mice.

[0143] Based on the prototype strain sequence, NTD and RBD were selected as candidate antigens. The immunogenicity of NTD alone, RBD alone, co-immunization with NTD and RBD, NTD-RBD fusion protein (hereinafter referred to as NR), and NTD-RBD-foldon fusion protein (hereinafter referred to as NR-foldon) was detected, and the results are shown in Table 12. The results showed that immunization of BALB / c mice with NR-foldon antigen and AS03 adjuvant significantly and effectively induced the production of high-titer neutralizing antibodies (based on the SARS-CoV-2 virus strain and VSV pseudovirus detection system) and antigen-specific IgG antibodies. The neutralizing effect against the prototype strain and D614G and the Brazilian mutant strain P.1(Gamma) was significantly better than that against other antigens.

[0144] In addition, even after the dosage of each antigen component in the RBD+NTD group was halved, a certain level of neutralizing antibodies and IgG antibodies could still be produced, indicating that this combination method itself also has good immune potential.

[0145] Table 11 Overview of Experimental Design for Recombinant SARS-CoV-2 Vaccine Candidate Antigens in BALB / c Mice

[0146]

[0147] Table 12 Screening results of recombinant COVID-19 vaccine candidate antigens in BALB / c mice

[0148]

[0149]

[0150] Note: 1. Detection of neutralizing antibodies against the novel coronavirus based on the SARS-CoV-2 strain, the same applies below.

[0151] 2. Pseudovirus types: original strain, major circulating strain (D614G); VOC variant strains: UK Alpha mutant strain (Alpha, B.1.1.7), Beta South African mutant strain (Beta, B.1.351), Gamma Brazilian mutant strain (Gamma, P.1), Delta Indian mutant strain (Delta, B.1.617.2); VOI variant strains: Lambda Peruvian mutant strain (Lambda, C.37), the same below.

[0152] Example 19: Construction of Vector and Protein Expression

[0153] Referring to Examples 1-4, the target fragment to be expressed was constructed as follows, and the protein was expressed and purified:

[0154] RBD(Alpha)-HBsAg-6*HIS; NTD(Alpha)-HBsAg-6*HIS; RBD(Alpha)-P2-6*HIS; NTD(Alpha)-P2-6*HIS; RBD(Beta)-HBsAg-6*HIS; NTD(Beta)-HBsAg-6*HIS; RBD(Beta)-P2-6*HIS; NTD(Beta)-P2-6*HIS; RBD(Gamma)-HBsAg-6*HIS; NTD(Gamma)-HBsAg-6*HIS; RB D(Gamma)-P2-6*HIS; NTD(Gamma)-P2-6*HIS; RBD(Delta)-HBsAg-6*HIS; NTD(Delta)-HBsAg-6*HIS; RBD(Delta)-P2-6*HIS; NTD( Delta)-P2-6*HIS; RBD(Omicron)-HBsAg-6*HIS; NTD(Omicron)-HBsAg-6*HIS; RBD(Omicron)-P2-6*HIS; NTD(Omicron)-P2-6*HIS

[0155] Example 20 Preparation of Pharmaceutical Composition

[0156] 0.02 mg / mL of RBD (prototype) protein, 0.02 mg / mL of NTD (prototype) protein, 0.02 mg / mL of RBD (Beta) protein, 0.02 mg / mL of NTD (Beta) protein; 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; and 5 mM to 25 mM histidine buffer at pH 5.0 to 7.0 were aliquoted into 2 ml control bottles, 0.5 ml per bottle, and lyophilized.

[0157] Example 21 Vaccine Preparation

[0158] The pharmaceutical composition described in Example 20 contains aluminum hydroxide adjuvant and CpG1018 adjuvant, wherein the final concentration of aluminum hydroxide adjuvant is 1 mg / mL and the final concentration of CpG1018 adjuvant is 6 mg / mL.

[0159] Example 22 Vaccine Preparation

[0160] The pharmaceutical composition described in Example 20 contains the following adjuvants: each adjuvant vial is 0.25 ml and includes 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of Tween 80, 3.53 mg of NaCl, 0.09 mg of KCl, 0.51 mg of Na2HPO4, 0.09 mg of KH2PO4, and water for injection.

[0161] Example 23 Preparation of pharmaceutical composition

[0162] 0.02 mg / mL of RBD (prototype) protein, 0.02 mg / mL of NTD (prototype) protein, 0.02 mg / mL of RBD (Omicron) protein, 0.02 mg / mL of NTD (Omicron) protein; 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; and 5 mM to 25 mM histidine buffer at pH 5.0 to 7.0 were aliquoted into 2 ml control bottles, 0.5 ml per bottle, and lyophilized.

[0163] Example 24 Vaccine Preparation

[0164] In Example 23, aluminum hydroxide adjuvant and CpG1018 adjuvant were added to the pharmaceutical composition, wherein the final concentration of aluminum hydroxide adjuvant was 1 mg / mL and the final concentration of CpG1018 adjuvant was 6 mg / mL.

[0165] Example 25: Preparation of the vaccine

[0166] The pharmaceutical composition described in Example 23 contains the following adjuvants: each adjuvant vial contains 0.25 ml and includes 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of Tween 80, 3.53 mg of NaCl, 0.09 mg of KCl, 0.51 mg of Na2HPO4, 0.09 mg of KH2PO4, and water for injection.

[0167] Example 26 Preparation of Pharmaceutical Composition

[0168] 0.02 mg / mL of RBD (Beta) protein, 0.02 mg / mL of NTD (Beta) protein, 0.02 mg / mL of RBD (Gamma) protein, 0.02 mg / mL of NTD (Gamma) protein; 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; and 5 mM to 25 mM histidine buffer at pH 5.0 to 7.0 were aliquoted into 2 ml control bottles, 0.5 ml per bottle, and lyophilized.

[0169] Example 27 Vaccine Preparation

[0170] The pharmaceutical composition described in Example 26 contains aluminum hydroxide adjuvant and CpG1018 adjuvant, wherein the final concentration of aluminum hydroxide adjuvant is 1 mg / mL and the final concentration of CpG1018 adjuvant is 6 mg / mL.

[0171] Example 28 Vaccine Preparation

[0172] The pharmaceutical composition described in Example 26 contains the following adjuvants: each adjuvant vial contains 0.25 ml and includes 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of Tween 80, 3.53 mg of NaCl, 0.09 mg of KCl, 0.51 mg of Na2HPO4, 0.09 mg of KH2PO4, and water for injection.

[0173] Example 29 Preparation of Pharmaceutical Composition

[0174] 0.02 mg / mL of RBD (Delta) protein, 0.02 mg / mL of NTD (Delta) protein, 0.02 mg / mL of RBD (Omicron) protein, 0.02 mg / mL of NTD (Omicron) protein; 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; and 5 mM to 25 mM histidine buffer at pH 5.0 to 7.0 were aliquoted into 2 ml control bottles, 0.5 ml per bottle, and lyophilized.

[0175] Example 30: Preparation of the vaccine

[0176] The pharmaceutical composition described in Example 29 contains aluminum hydroxide adjuvant and CpG1018 adjuvant, wherein the final concentration of aluminum hydroxide adjuvant is 1 mg / mL and the final concentration of CpG1018 adjuvant is 6 mg / mL.

[0177] Example 31 Vaccine Preparation

[0178] The pharmaceutical composition described in Example 29 contains the following adjuvants: each adjuvant vial contains 0.25 ml and includes 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of Tween 80, 3.53 mg of NaCl, 0.09 mg of KCl, 0.51 mg of Na2HPO4, 0.09 mg of KH2PO4, and water for injection.

[0179] Example 32: Detection of the effect of recombinant COVID-19 vaccine candidate antigen on inducing neutralizing antibody production in mice.

[0180] The vaccines provided in Examples 12, 22, 25, 28, and 31 were used as candidate vaccines, and mice were immunized according to the method provided in Example 18. The immunogenicity of each group of vaccines against different strains of pseudoviruses was compared, and the results are shown in Table 14.

[0181] The results showed that all groups of vaccines could produce a certain level of neutralizing antibodies and a high level of antigen-specific IgG antibodies against different strains, and the level of neutralizing antibodies produced against the corresponding strains was generally higher than that against other strains. The overall effect of the bivalent vaccines was better than that of the monovalent vaccines. Among them, the original strain + Omicron group (Example 25) produced the highest level of neutralizing antibodies overall, indicating that all groups of vaccines could play a certain role in immunity against different strains, with the original strain + Omicron being more prominent. In particular, it has certain application potential against the highly infectious Omicron strain.

[0182] Table 13 Overview of Experimental Design for Recombinant SARS-CoV-2 Vaccine Candidate Antigens in BALB / c Mice

[0183]

[0184] Table 14 Screening results of recombinant COVID-19 vaccine candidate antigens in BALB / c mice

[0185]

[0186] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An immunogenic composition, characterized in that, The product comprises a first component and a second component. The first component is formed by mixing the receptor-binding domains (RBDs) of the spike proteins (S proteins) of two novel coronavirus strains: the amino acid sequence of the prototype strain RBD is shown in SEQ ID NO:1, and the amino acid sequence of the Omicron BA.1 strain RBD is shown in SEQ ID NO:

5. The second component is formed by mixing the N-terminal domains (NTDs) of the S proteins of two novel coronavirus strains: the amino acid sequence of the prototype strain NTD is shown in SEQ ID NO:9, and the amino acid sequence of the Omicron BA.1 strain NTD is shown in SEQ ID NO:

13. The weight ratio of the first component to the second component is 1:

1. In the first component, the weight ratio of the two RBDs is 1:

1. In the second component, the weight ratio of the two NTDs is 1:

1.

2. The immunogenic composition according to claim 1, characterized in that, The RBD in the first component and the NTD in the second component are each linked to a polypeptide: P2 or its functional active fragment, the amino acid sequence of which is shown in any one of SEQ ID NO:17-19.

3. The immunogenic composition according to claim 2, characterized in that, The polypeptide is directly or indirectly linked to the RBD, and the polypeptide is directly or indirectly linked to the NTD.

4. The immunogenic composition according to claim 3, characterized in that, The polypeptide is fused with the RBD within the frame, and the polypeptide is fused with the NTD within the frame.

5. The immunogenic composition according to claim 3, characterized in that, The direct or indirect connection includes connection via connectors.

6. The immunogenic composition according to claim 5, characterized in that, The connector can be a rigid joint or a flexible joint.

7. The immunogenic composition according to claim 6, characterized in that, The amino acid sequence of the linker is shown in any one of SEQ ID NO:27-28.

8. A pharmaceutical composition comprising the immunogenic composition of any one of claims 1-7, and a pharmaceutically acceptable excipient.

9. Use of the immunogenic composition of any one of claims 1-7, or the pharmaceutical composition of claim 8, in the preparation of a vaccine for the prevention of novel coronavirus infection.

10. A method for preparing a subunit vaccine for the prevention of novel coronavirus infection, comprising: 1) Provide the immunogenic composition according to any one of claims 1-7 or the pharmaceutical composition according to claim 8; as well as 2) Mix the immunogenic composition or pharmaceutical composition described in 1) with a pharmaceutically acceptable adjuvant.

11. A subunit vaccine for preventing novel coronavirus infection prepared according to the method of claim 10.

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