Novel coronavirus fusion protein vaccine and application thereof

Abstract

The present disclosure provides a novel coronavirus fusion protein vaccine and application thereof. The vaccine comprises a recombinant fusion protein, a buffer, a stabilizer, a surfactant, sodium chloride and an adjuvant, wherein the recombinant fusion protein comprises an interferon or a functional fragment thereof, a novel coronavirus SARS-CoV-2 or a functional fragment thereof and IgG1 Fc. The concentration of sodium chloride in the vaccine is maintained in a suitable range, which does not negatively affect the stability of the vaccine, and can ensure a good adsorption rate of the aluminum adjuvant. The vaccine of the present disclosure has good high temperature, oscillation and freeze-thaw stability.

Description

Technical Field

[0001] This disclosure relates to the fields of epidemic prevention and vaccine production, specifically to the application of a novel coronavirus fusion protein vaccine. Background Technology

[0002] The novel coronavirus is composed of four structural proteins (spike glycoprotein, envelope protein, membrane protein, and nucleocapsid protein) and an RNA nucleic acid chain. The spike glycoprotein (S protein) is a glycoprotein located on the surface of the SARS-CoV-2 membrane and primarily functions in cell adhesion and cell membrane fusion. The S protein consists of two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain (RBD), which is responsible for recognizing the host cell receptor ACE2. This is a key factor in virus-receptor interaction and viral invasion of cells, and also a key target for vaccine design.

[0003] COVID-19 vaccines are an effective and economical way to eliminate the SARS-CoV-2 virus. Several vaccines are currently available, providing protection for human health. Recombinant protein vaccines are characterized by stable production, easy scale-up, ease of storage and transportation, and low cost. However, recombinant proteins are sensitive to conditions such as protein concentration, pH, excipients, and repeated freeze-thaw cycles, which can lead to reduced purity and / or decreased activity. Therefore, each protein needs to be stored and used under suitable conditions to ensure stability. Typically, when a single protein is used as an antigenic component of a vaccine, its immunogenicity is weak, requiring the addition of adjuvants to enhance the vaccine's effectiveness.

[0004] Therefore, further research and exploration are needed to find an effective and safe COVID-19 vaccine. Summary of the Invention

[0005] In order to address the problems existing in the prior art, the purpose of this disclosure is to provide a new, highly effective and safe recombinant novel coronavirus fusion protein vaccine.

[0006] To achieve the above objectives, this disclosure adopts the following specific solutions:

[0007] In one aspect, this disclosure relates to a recombinant novel coronavirus (SARS-CoV-2) fusion protein vaccine comprising: a recombinant fusion protein, a buffer solution, a stabilizer, a surfactant, sodium chloride, and an adjuvant, wherein the recombinant fusion protein comprises interferon or a functional fragment thereof, novel coronavirus SARS-CoV-2 or a functional fragment thereof, and IgG1 Fc.

[0008] In some embodiments of this disclosure, the amino acid sequence of the fusion protein is shown in any of SEQ ID NO 1-7.

[0009] In another aspect, this disclosure relates to the use of a recombinant novel coronavirus (SARS-CoV-2) fusion protein vaccine in the preparation of a medicament for the prevention and / or treatment of novel coronavirus SARS-CoV-2 infection and / or novel coronavirus disease COVID-19.

[0010] In another aspect, this disclosure relates to a method for preventing and / or treating SARS-CoV-2 infection and / or COVID-19, comprising administering an effective amount of the aforementioned vaccine to a subject.

[0011] On the other hand, this disclosure relates to a method for inducing a neutralizing antigen-specific immune response in an individual, comprising administering the aforementioned vaccine to a subject.

[0012] A major highlight of the recombinant novel coronavirus fusion protein vaccine disclosed herein (such as the V-01 recombinant fusion protein vaccine) is its safety and protective efficacy, with a significant advantage in protecting the elderly and those with underlying diseases. According to data from the sequential booster Phase III clinical trial of V-01, the incidence of systemic adverse reactions (fever, headache, and muscle pain, etc.) in the V-01 trial was superior to similar booster trials using mRNA vaccines or adenovirus vector vaccines.

[0013] One reason for the good safety and protective efficacy of the recombinant novel coronavirus fusion protein vaccine disclosed herein is its molecular design: the C-terminus of recombinant human IFNα-2b is linked to the N-terminus of RBD via a linker (containing a Pan epitope sequence), and then the C-terminus of RBD is linked to human IgG1 Fc, forming a symmetrical bivalent Fc fusion protein structure. The advantage of this molecular design is that the fusion expression of RBD and human IgG1 Fc forms a stable dimer structure, overcoming the weakness of weak immunogenicity in monomeric RBD protein vaccines. The fusion of human IFNα-2b into the N-segment of RBD forms the unique recombinant fusion protein vaccine IFN-RBD-Fc, improving the efficiency of dendritic cell (DC) antigen capture and presentation, and promoting vaccine antibody and T cell responses. Specifically, the V-01 design innovatively incorporates human interferon as a biological adjuvant.

[0014] The second reason why the recombinant novel coronavirus fusion protein vaccine disclosed herein exhibits good safety and protective efficacy is the effectiveness of conventional aluminum adjuvant. Given that the safety of traditional aluminum adjuvants has been verified, the recombinant novel coronavirus fusion protein vaccine also incorporates conventional aluminum adjuvant, circumventing the production and safety issues associated with novel adjuvants.

[0015] To address the complexity and instability of recombinant novel coronavirus fusion protein molecules, the inventors first explored the pH and stabilizers needed to stabilize the protein, and then studied the conditions for complete adsorption of the stable protein with aluminum adjuvant. Ultimately, a stable recombinant novel coronavirus fusion protein vaccine was provided. The vaccine is produced from Chinese hamster ovary (CHO) cells that efficiently express the recombinant novel coronavirus fusion protein. The stock solution of the vaccine is obtained through cell culture, clarification filtration, and high purification. This stock solution is then diluted with a diluent, mixed with aluminum hydroxide adjuvant, and packaged to obtain the final product. The finished product can be stored and transported for extended periods at 2-8℃, exhibits good stability after 6 months of accelerated adsorption at 25±2℃, and remains stable after 1 month of intensive accelerated adsorption at 40±2℃. The recombinant novel coronavirus fusion protein vaccine features stable production, easy scale-up, and low cost. Attached Figure Description

[0016] Figure 1 Antibody titers in mice immunized with different amounts of aluminum adjuvant 14 days later

[0017] Figure 2 Adsorption rate DOE contour plot

[0018] Figure 3 DOE purity contour plot

[0019] Figure 4 Effect of different aluminum adjuvant contents on the maximum adsorption capacity of V-01 protein Detailed Implementation

[0020] Experimental methods in the following embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical and biological reagents used in the embodiments are commercially available products.

[0021] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure.

[0022] The terms “comprising” and “having”, and any variations thereof, in this disclosure are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0023] In this disclosure, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship. Furthermore, to better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0024] As used in this disclosure, the term "RBD" stands for receptor-binding domain, which is a key region for ARS-CoV-2 to bind to the ACE2 receptor and for antibody recognition.

[0025] The terms “Chinese hamster ovary cells” or “CHO cells” used in this disclosure are the most representative mammalian expression vectors in genetically engineered vaccine research and are also the most commonly used type of cells to express exogenous proteins.

[0026] As used in this disclosure, the term "adjuvant," also known as an immunomodulator or immunostimulant, is an additive to vaccines that, when injected into the body before or mixed with the antigen, enhances the body's immune response to the antigen or alters the type of immune response. It is a non-specific immunostimulant and is not antigenic itself. An ideal adjuvant not only enhances the immune response but also enables the body to acquire optimal protective immunity.

[0027] As used in this disclosure, the term "antigen" refers to a substance, typically a protein, capable of inducing an immune response in a subject. The term also refers to an immunologically active protein, i.e., a protein that, once administered to a subject (either directly or by administering to the subject a nucleotide sequence or carrier encoding the protein), is capable of eliciting a humoral and / or cellular immune response against that protein.

[0028] In one aspect, this disclosure relates to a novel coronavirus (SARS-CoV-2) recombinant fusion protein vaccine comprising: a recombinant fusion protein, a buffer solution, a stabilizer, a surfactant, sodium chloride, and an adjuvant, wherein the recombinant fusion protein comprises interferon or a functional fragment thereof, novel coronavirus SARS-CoV-2 or a functional fragment thereof, and IgG1 Fc.

[0029] In some embodiments of this disclosure, the functional fragment of SARS-CoV-2 is its receptor-binding domain (RBD).

[0030] In some embodiments of this disclosure, the pH of the vaccine is 4.0-5.0. In some preferred embodiments of this disclosure, the pH is 4.3-4.9.

[0031] In some embodiments of this disclosure, the recombinant fusion protein comprises an amino acid sequence having 80% or more identity with an amino acid sequence selected from any of SEQ ID NO 1-7, preferably having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably having 98% or 99% or more identity; preferably, the amino acid sequence of the fusion protein is as shown in any of SEQ ID NO 1-7.

[0032] In some embodiments of this disclosure, the concentration of the recombinant fusion protein in the recombinant fusion protein vaccine is 0.001-0.9 mg / mL, preferably 0.01-0.9 mg / mL.

[0033] In some embodiments of this disclosure, the buffer solution is one or more of acetate buffer, citrate buffer, glutamate buffer, glycine-hydrochloric acid buffer, and disodium hydrogen phosphate-citrate buffer.

[0034] In some embodiments of this disclosure, the concentration of the buffer solution is 5-80 mM, preferably 18-22 mM.

[0035] In some embodiments of this disclosure, the buffer solution is an acetate-sodium acetate buffer solution.

[0036] In some embodiments of this disclosure, the stabilizer is selected from one or more of sucrose, trehalose, mannitol, sorbitol, lactose, glucose, maltose, galactose, fructose, sorbitol, raffinose, glucosamine, N-methylglucosamine, galactosamine, neuraminic acid, and amino acids. Preferably, the amino acid is selected from one or more of arginine, methionine, glycine, lysine, histidine, and proline.

[0037] In some embodiments of this disclosure, the stabilizer is sucrose; more preferably, the concentration of sucrose is 1%-10%, preferably 4%-6%.

[0038] In some embodiments of this disclosure, trehalose is used as a stabilizer; more preferably, the concentration of trehalose is 1%-10%, and more preferably 4%-6%.

[0039] In some embodiments of this disclosure, the amino acid is glycine; more preferably, the concentration of glycine is 1-10 mg / mL, and more preferably 3.5-7.5 mg / mL.

[0040] In some embodiments of this disclosure, the surfactant is polysorbate, polyoxyethylene alkyl ether (benzyl ether), alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymer (poloxam, prolonic acid), and sodium dodecyl sulfate (SDS).

[0041] In some embodiments of this disclosure, the polysorbate is a polyoxyethylene dehydrated sorbitol fatty acid ester (Tween), more preferably polysorbate 20 or polysorbate 80.

[0042] In some embodiments of this disclosure, the concentration of the surfactant is 0.01-2 mg / mL; more preferably 0.1-0.3 mg / mL.

[0043] In some embodiments of this disclosure, the concentration of sodium chloride is 60-120 mM, preferably 70-90 mM.

[0044] In some embodiments of this disclosure, the adjuvant is selected from aluminum adjuvants, monophosphoryl lipid A or derivatives thereof, oil-in-water emulsion adjuvants, liposome adjuvants, unmethylated CpG containing oligonucleotides, and combinations thereof.

[0045] In some embodiments of this disclosure, the oil-in-water emulsion adjuvant contains 3D MPL, QS21, and tocopherol.

[0046] In some embodiments of this disclosure, the oil-in-water emulsion adjuvant contains Tween 80, Span 85, and squalene.

[0047] In some embodiments of this disclosure, the oil-in-water emulsion adjuvant contains Tween 80, squalene, and tocopherol.

[0048] In some embodiments of this disclosure, the liposome adjuvant contains 3D MPL and QS21.

[0049] In some embodiments of this disclosure, the adjuvant comprises a combination of unmethylated CpG of oligonucleotides and an aluminum adjuvant.

[0050] In some embodiments of this disclosure, the aluminum adjuvant is selected from aluminum hydroxide (Al(OH)3), aluminum phosphate (AlPO4), aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate (AAHS) or alum (KAl(SO4)·12H2O).

[0051] In some preferred embodiments of this disclosure, the aluminum adjuvant is aluminum hydroxide.

[0052] In some embodiments of this disclosure, the concentration of aluminum hydroxide is 0.125-0.625 mg / mL.

[0053] In some preferred embodiments of this disclosure, the concentration of aluminum hydroxide is 0.25-0.6 mg / mL. In some preferred embodiments of this disclosure, the concentration of aluminum hydroxide is 0.4-0.6 mg / mL; preferably 0.5 mg / mL.

[0054] In another aspect, this disclosure relates to the use of a recombinant novel coronavirus (SARS-CoV-2) fusion protein vaccine in the preparation of a medicament for the prevention and / or treatment of novel coronavirus SARS-CoV-2 infection and / or novel coronavirus disease COVID-19.

[0055] In another aspect, this disclosure relates to a method for preventing and / or treating SARS-CoV-2 infection and / or COVID-19, comprising administering an effective amount of the aforementioned vaccine to a subject.

[0056] On the other hand, this disclosure relates to a method for inducing a neutralizing antigen-specific immune response in an individual, comprising administering the aforementioned vaccine to a subject.

[0057] The vaccine disclosed herein has the following main advantages:

[0058] (1) This disclosure found that the presence of sodium chloride is detrimental to the stability of the novel coronavirus fusion protein, but the adsorption capacity of aluminum adjuvant is weak in the sodium chloride-free system. Therefore, it was determined that the stock solution formulation does not contain sodium chloride, while the formulation contains sodium chloride. Further research by the applicant found that adding a certain amount of sodium chloride is beneficial to improving the adsorption of aluminum adjuvant to antigen protein. When the sodium chloride content is 70-90 mM, it can maximize the adsorption rate of aluminum adjuvant and minimize the interference of sodium chloride on protein stability.

[0059] (2) This disclosure found that when the pH of the vaccine is maintained at 4-5, the novel coronavirus fusion protein (e.g., V-01 recombinant fusion protein) has good stability; when the pH is greater than 5, the recombinant fusion protein may flocculate and precipitate significantly.

[0060] (3) The vaccine provided in this disclosure performed well in accelerated stability, strong accelerated stability and oscillation stability tests, and has high robustness.

[0061] Example

[0062] The technical solutions of this disclosure are further illustrated below through specific implementation methods. Those skilled in the art should understand that the embodiments described are merely illustrative of this disclosure and should not be construed as specific limitations thereof.

[0063] Example 1: Preparation of Recombinant Novel Coronavirus Fusion Protein Vaccine Stock Solution

[0064] The recombinant novel coronavirus fusion protein disclosed herein (e.g., V-01 recombinant fusion protein) is formed by linking the C-terminus of recombinant human IFNα-2b to the N-terminus of the SARS-CoV-2 spike protein RBD via a linker (containing a Pan epitope sequence), and then linking the C-terminus of the RBD to human IgG1 Fc, forming a symmetrical bivalent Fc fusion protein structure. The recombinant novel coronavirus fusion protein is the active ingredient of the vaccine. The recombinant novel coronavirus fusion protein is produced by a Chinese hamster ovary (CHO) cell expression vector that efficiently expresses the recombinant novel coronavirus fusion protein. The stock solution of the recombinant novel coronavirus fusion protein vaccine is obtained after CHO cell culture, clarification and filtration of the cell culture medium, and high purification.

[0065] The amino acid sequence of the V-01-recombinant fusion protein (SEQ ID NO.1) is as follows:

[0066] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRS KEGGGGSGGGGSGGGGSAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVG GNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0067] Other recombinant novel coronavirus fusion protein vaccines (such as the South African strain vaccine (SEQ ID NO.2), the delta strain vaccine (SEQ ID NO.3), the Omicron strain vaccine 529 (SEQ ID NO.4), the Omicron strain vaccine 529-11 (SEQ ID NO.5), the Omicron strain vaccine 529-12 (SEQ ID NO.6), and the Omicron broad-spectrum vaccine (B5) (SEQ ID NO.7)) have the same active ingredient structure as the V-01 recombinant fusion protein, differing only in their RBD. The same method can be used to obtain the corresponding recombinant novel coronavirus fusion protein vaccine stock solution. Their amino acid sequences are as follows:

[0068] The amino acid sequence of the South African strain vaccine (SEQ ID NO.2) is as follows:

[0069] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRS KEGGGGSGGGGSGGGGSAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVG GNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0070] The amino acid sequence of the delta strain vaccine (SEQ ID NO.3) is as follows:

[0071] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFEPKSCDKTHTCPPCPAPELLGGGPSPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0072] The amino acid sequence of the Omicron strain vaccine 529 (SEQ ID NO.4) is as follows:

[0073] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAW

[0074] DETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFS

[0075] LSTNLQESLRSKEGGGGSGGGGSGGGGSAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFDEVFNATRF

[0076] ASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNY

[0077] KLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLKSYSFRP

[0078] TYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

[0079] QLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN

[0080] KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0081] GSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0082] The amino acid sequence of the Omicron strain vaccine 529-11 (SEQ ID NO.5) is as follows:

[0083] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAW

[0084] DETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFS

[0085] LSTNLQESLRSKEGGGGSGGGGSGGGGSAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRF

[0086] ASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNY

[0087] KLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRP

[0088] TYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

[0089] QLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN

[0090] KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0091] GSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0092] The amino acid sequence of the Omicron strain vaccine 529-12 (SEQ ID NO.6) is as follows:

[0093] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAW

[0094] DETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFS

[0095] LSTNLQESLRSKEGGGGSGGGGSGGGGSAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRF

[0096] ASVYAWNRKRISNCVADYSVLYNLASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNY

[0097] KLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRP

[0098] TYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

[0099] QLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN

[0100] KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0101] GSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0102] The amino acid sequence of the Omicron broad-spectrum vaccine (B5) (SEQ ID NO.7) is as follows:

[0103] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAW

[0104] DETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFS

[0105] LSTNLQESLRSKEGGGGSGGGGSGGGGSAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATKF

[0106] ASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNY

[0107] KLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLRSYGFRP

[0108] TYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

[0109] QLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN

[0110] KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0111] GSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0112] Example 2: pH Screening of Recombinant Novel Coronavirus Fusion Protein Vaccine Formulation

[0113] pH is a key factor affecting protein stability. In this embodiment, the optimal pH range of the V-01 recombinant fusion protein vaccine formulation was determined through two rounds of screening.

[0114] 1. Initial pH screening (Liq2, first round)

[0115] Experimental objective: To conduct an initial large-scale pH screening. Since common protein formulations have a pH range of 5-8, and phosphate buffer pairs have buffering capacity within this range, the first round of screening used phosphate buffer pairs to adjust the pH of recombinant fusion protein vaccine formulations to a suitable level.

[0116] Experimental Methods: Based on 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate, 60 mg / mL sucrose, and 0.2 mg / mL polysorbate-80, V-01 recombinant fusion protein formulations were prepared at pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, and pH 7.5, respectively. The thermal stability and purity of each experimental group were investigated (observed and tested by DSC and SEC-HPLC). The experimental design for different formulations is shown in Table 1.

[0117] Table 1. pH screening prescription design (Liq2)

[0118]

[0119]

[0120] Experimental results:

[0121] (1) The thermal stability (DSC) test data of different experimental groups are shown in Table 1. There was no significant difference in thermal stability of recombinant protein preparations at different pH values. Tonset was at 43℃, Tm1 at 52℃, Tm2 at 59℃, and Tm3 at 83℃.

[0122] (2) The purity (SEC-HPLC) test data of different experimental groups are shown in Table 2. No monomer peaks were observed in the samples at pH 5.0 and pH 5.5. The monomer content of the samples at pH 6.0, pH 6.5, pH 7.0, and pH 7.5 was much lower than that of the purified intermediate (approximately 90%). Simultaneously, during the purification process, it was found that when the pH of the affinity chromatography sample (approximately pH 3.9) was gradually increased back to pH 5.0 and then further increased, the sample flocculated and precipitated significantly. This indicates that V-01 protein is more suitable for stable existence in a slightly acidic environment. Therefore, the pH value of V-01 protein needs to be maintained at pH 5.0 or below.

[0123] Referring to marketed biological products Brodalumab (subcutaneous injection, pH 4.8), Cimzia (subcutaneous injection, pH 4.7), and Neupogen (subcutaneous or intravenous injection, pH 4.0), the pH range for further exploration of the V-01 formulation is pH 4.0 to pH 5.0.

[0124] Table 2. pH screening DSC thermal stability results (Liq2)

[0125] prescription Tonset Tm1 Tm2 Tm3 F2 pH5.0 43.4 51.8 59.2 83.3 F2 pH 5.5 45.2 51.9 59.1 83.0 F3 pH 6.0 45.3 52.4 59.2 83.4 F4 pH 6.5 45.8 52.4 59.2 83.1 F5 pH 7.0 45.9 52.1 59.1 82.8 F6 pH 7.5 45.3 52.1 59.2 82.2

[0126] Table 3. pH screening SEC-HPLC purity results (Liq2)

[0127] experimental group Monomer percentage (%) Polymer percentage (%) Proportion of low molecular weight substances (%) V-01Liq2 F1 pH5.0 at zero time 0 59.9 40.1 V-01Liq2 F2 pH5.5 at zero time 0 85.1 14.9 V-01Liq2 F3 pH6.0 at zero time 40.6 50.8 8.6 V-01Liq2 F4 pH6.5 at 0 69.7 27.7 2.6 V-01Liq2 F5 pH7.0 at zero time 73.1 21.8 5.1 V-01Liq2 F6 pH7.5 at zero time 37.9 20.3 41.8

[0128] 2.2 pH rescreening (Liq5, second round)

[0129] Objective: To study the stability of V-01 protein in the pH range of 4.0 to 5.0.

[0130] Experimental Methods: The acetate-sodium acetate buffer pair exhibits good buffering capacity in the pH range of 4.0–5.0. V-01 recombinant fusion protein vaccine formulations at pH 4.0, pH 4.5, and pH 5.0 were prepared based on 20 mM acetate-sodium acetate, 60 mg / mL sucrose, 7.5 mg / mL glycine, and 0.2 mg / mL polysorbate-80. The stability of the protein at different pH levels was evaluated using a purified intermediate (50 mM acetate-sodium acetate, 330 mM sodium chloride, pH 5.0) as a control. The formulation design is shown in Table 4.

[0131] Table 4 pH screening prescription design (Liq5)

[0132]

[0133] Experimental results:

[0134] Table 4 shows the SEC-HPLC purity test data for different experimental groups. The zero-time monomer samples in the pH 4.0, pH 4.5, and pH 5.0 groups all had a purity greater than 88%, indicating that the recombinant protein formulation has good stability at pH 4.0-5.0. This is also higher than that of the purified intermediate (control group), indicating that the excipients (sucrose + glycine) provide better protection for the protein. After 7 days of accelerated testing at 30℃, the purity of the pH 4.0 and pH 4.5 samples showed no significant difference compared to the zero-time values, while the purity of the pH 5.0 sample was slightly lower than that of the zero-time values. In conclusion, the V-01 recombinant fusion protein formulation exhibits good stability at pH 4.0-pH 5.0.

[0135] In particular, the monomer purity of the control group decreased significantly after 7 days, indicating that the presence of sodium chloride may be detrimental to the long-term stability of V-01 protein, and the concentration of sodium chloride needs to be controlled.

[0136] Table 5. pH screening SEC-HPLC purity results (Liq5)

[0137]

[0138] Example 3: Screening of adjuvant content in recombinant novel coronavirus fusion protein vaccine formulation

[0139] The use of adjuvants can significantly enhance the level of vaccine immune response. Traditional aluminum hydroxide adjuvant has shown a strong level of immune response and has the longest history of safe use. In this embodiment, aluminum hydroxide is selected as an adjuvant to enhance the immune response of the vaccine.

[0140] In the case of an equal volume of V-01 recombinant fusion protein formulation (4 μg / dose), 6–8 week old C57BL / 6 mice were immunized with test samples containing different doses of aluminum adjuvant (0.1 mg / dose, 0.05 mg / dose, 0.02 mg / dose, 0 mg / dose or 0.625 mg aluminum adjuvant / 25 μg antigen, 0.3125 mg aluminum adjuvant / 25 μg antigen, 0.125 mg aluminum adjuvant / 25 μg antigen, 0 mg aluminum adjuvant / 25 μg antigen) to investigate the effect of different doses of aluminum adjuvant on the immunogenicity of mice. Each mouse was injected intramuscularly with 0.1 mL of serum in the thigh. Each group consisted of 17 mice. After 14 days, blood was collected from the orbital sinus. The blood was allowed to stand at room temperature until it coagulated, then centrifuged at 4000 rpm at 2–8°C for 10 min. The supernatant was collected, and the geometric mean titer (GMT) of the anti-SARS-CoV-2 RBD antibody was determined using an enzyme-linked immunosorbent assay kit (Acro, catalog number SPD-C52H3) to compare immunogenicity.

[0141] The antibody titer results of mice immunized with different aluminum adjuvant concentrations 14 days later are shown in the figure. Figure 1 Compared with the blank group without adjuvant, aluminum adjuvant doses of 20-100 μg / dose increased the immune effect. In the experimental group with adjuvant, the antibody titer increased with the increase of adjuvant concentration, and the effect basically reached saturation after exceeding 50 μg / dose.

[0142] Therefore, when the aluminum adjuvant content is 0.125-0.625 mg / 25 μg antigen, it can enhance the intensity and duration of the immune response, with 0.25-0.5 mg / 25 μg antigen being the preferred value.

[0143] Example 4: Study on the effect of each component in the formulation of the recombinant novel coronavirus fusion protein vaccine on the adsorption capacity of aluminum adjuvant.

[0144] It is necessary to clarify the contribution of each component in the formulation that maintains the stability of the V-01 recombinant fusion protein to the adsorption capacity of aluminum adjuvant. Therefore, each component was validated separately.

[0145] 1. Effects of each component of the formulation on the adsorption capacity of aluminum adjuvant

[0146] The formulation design is shown in Table 5. Formulations F1-F5 from Table 6 were mixed with aluminum adjuvant at a volume ratio of 1:1 for adsorption as the experimental group. Formulations F1-F5 from Table 6 were mixed with their corresponding buffer solutions at a volume ratio of 1:1 as the control group. The absorbance of the supernatant after adsorption in each experimental group was measured at 280 nm using a UV detector, and the adsorption rate was calculated. Adsorption rate = (Absorbance of unadsorbed protein - Absorbance of adsorbed protein) / Absorbance of unadsorbed protein × 100%.

[0147] Table 6 V-01Liq6 Prescription Composition

[0148]

[0149]

[0150] The experimental results are shown in Table 6. The adsorption capacity of each component of formulations F1-F4 combined with aluminum adjuvant in the experimental groups was relatively weak. However, the adsorption capacity of sodium chloride combined with aluminum adjuvant in formulation F5 was very high, reaching 95%. In summary, it is speculated that sodium chloride has a significant impact on the adsorption capacity of aluminum adjuvant.

[0151] Table 7 shows the adsorption rates of V-01 Liq6 formulation and aluminum adjuvant.

[0152] experimental group Adsorption supernatant A280 Adsorption rate Liq6 F1-Al 0.294 12％ Liq6 F1 - Control 0.334 N / A Liq6 F2-Al 0.289 8％ Liq6 F2 - Control 0.313 N / A Liq6 F3-Al 0.317 11％ Liq6 F3 - Control 0.357 N / A Liq6 F4-Al 0.274 14％ Liq6 F4 - Reference 0.317 N / A Liq6 F5-Al 0.021 95％ Liq6 F5 - Reference 0.376 N / A

[0153] Note: "N / A" indicates that there is no data here.

[0154] 2. Effect of sodium chloride on the adsorption capacity of aluminum adjuvants (Liq6)

[0155] To further verify the effect of sodium chloride on the adsorption capacity of aluminum adjuvant, “Liq6 FN-control” protein samples (where N represents the formulation number) were taken, and appropriate amounts of sodium chloride were added to make the protein samples contain 100mM, 300mM, and 500mM of sodium chloride, respectively. These samples were then mixed with aluminum adjuvant (or corresponding buffer solution) for adsorption. The absorbance of the supernatant after adsorption in each experimental group was detected at 280nm using a UV detector, and the adsorption rate was calculated.

[0156] The experimental results are shown in Table 7. The adsorption rates of both the formulation and aluminum adjuvant were significantly improved after the addition of sodium chloride. This further demonstrates that the addition of sodium chloride is beneficial to improving the adsorption rate of aluminum adjuvant.

[0157] Table 8. Results of adsorption rates of V-01 Liq6 with added sodium chloride and aluminum adjuvant.

[0158] experimental group Estimated concentration of added NaCl (mM) Adsorption supernatant A280 Adsorption rate Liq6 F1-Control + NaCl-Al 100 0.020 85％ Liq6 F1-control + NaCl-control 100 0.136 N / A Liq6 F2- control + NaCl- Al 300 0.024 84％ Liq6 F2- control + NaCl- control 300 0.149 N / A Liq6 F3-control + NaCl-Al 100 0.018 89％ Liq6 F3-control + NaCl-control 100 0.170 N / A Liq6 F4-control + NaCl-Al 500 0.018 89％ Liq6 F4-control + NaCl-control 500 0.166 N / A Liq6 F5-Control + NaCl-Al 82.5 0.022 83％ Liq6 F5-Control + NaCl-Control 82.5 0.127 N / A

[0159] Note: "N / A" indicates that there is no data here.

[0160] Example 5: Study on component concentration and conditions of recombinant novel coronavirus fusion protein vaccine formulation

[0161] The pH range and sodium chloride concentration in the recombinant fusion protein vaccine formulation were studied using a two-factor, three-level DOE design. The optimal conditions were explored within the pH range of 4.2-4.8 and sodium chloride concentration of 0-120 mM. The specific formulation design is shown in Table 9. Furthermore, considering that trehalose is more acid-resistant than sucrose under acidic pH conditions, the excipient sucrose was replaced with 4% trehalose. Simultaneously, to maintain the osmotic pressure of the finished product at a near-isotonic level, the contents of the excipients trehalose and glycine were appropriately reduced. The adsorption rate and SEC-HPLC purity of different formulation experimental groups were measured.

[0162] Table 9V-01 DOE Design Recipe (Liq14)

[0163]

[0164]

[0165] Adsorption rate data are shown in Table 10, and SEC-HPLC purity data are shown in Table 8.

[0166] Table 10V-01Liq14 Adsorption Rate Results

[0167]

[0168] Note: Adsorption rate = (Peak area of ​​SEC-HPLC purity of unadsorbed sample - Peak area of ​​SEC-HPLC purity of adsorbed sample) / Peak area of ​​SEC-HPLC purity of unadsorbed sample × 100%. For example, F1-Al adsorption rate = (638.2 - 7.9) / 638.2 * 100% = 99%.

[0169] Note: "N / A" indicates that there is no data here.

[0170] Table 11 V-01Liq14 SEC-HPLC Purity Results

[0171]

[0172]

[0173] Note: "LMW" indicates low molecular weight substances.

[0174] “ND” indicates that no result was detected.

[0175] Differences in purity between formulations were observed after acceleration at 30℃. The adsorption rate results and purity results after acceleration at 30℃ were input into MATLAB software for DOE analysis. Adsorption rate DOE contour plots and purity DOE contour plots were obtained, see [link to MATLAB software]. Figure 2 The DOE response surface regression analysis results for adsorption rate and purity are shown in Table 12.

[0176] Table 12-1V-01 Liq14 Adsorption Rate DOE Response Surface Regression Analysis

[0177] Factors affecting adsorption rate p-value analyze pH 0.050 The effect of pH on adsorption rate is at a critical value, and the impact cannot be assessed. NaCl 0.000 Sodium chloride concentration has a significant effect on adsorption rate. pH*pH 0.617 The square of pH has no significant effect on the adsorption rate. NaCl*NaCl 0.000 The square of sodium chloride concentration has a significant effect on the adsorption rate. pH*NaCl 0.066 The interaction between pH and sodium chloride concentration had no significant effect on the adsorption rate.

[0178] Table 12-2V-01 Liq14 Purity DOE Response Surface Regression Analysis

[0179] Factors affecting purity % p-value analyze pH 0.100 pH has no significant effect on purity. NaCl 0.003 Sodium chloride concentration has a significant impact on purity. pH*pH 0.342 The square of pH has no significant effect on purity. NaCl*NaCl 0.041 The square of sodium chloride concentration has a significant effect on purity. pH*NaCl 0.046 The interaction between pH and sodium chloride concentration has a significant impact on purity.

[0180] Note: A p-value greater than 0.05 indicates no significant effect. A p-value less than 0.05 indicates a significant effect.

[0181] Response surface regression analysis revealed that sodium chloride concentration significantly affects both protein purity and adsorption rate. The sodium chloride concentration needs to be maintained within a suitable range to ensure both the stability of the V-01 recombinant fusion protein vaccine formulation and a good adsorption rate.

[0182] The pH range within the study area had no significant effect on protein purity and adsorption rate, meaning the pH range of 4.2–4.8 is applicable. See also Figure 2 and Figure 3 According to the standard of a finished product adsorption rate of not less than 90%, a sodium chloride concentration of 60-120 mM is suitable. According to the standard requirement of a finished product adsorption rate of not less than 95% (to raise the quality standard), the sodium chloride concentration must be not less than 70 mM. Therefore, the center point of sodium chloride concentration in the finished product formulation is 80 mM ± 10 mM (operating margin). To ensure the purity and stability of the stock solution, the stock solution formulation does not contain sodium chloride.

[0183] Example 6: Study on the maximum adsorption capacity of aluminum adjuvant for the relevant antigen (V-01)

[0184] Based on existing conclusions, the maximum adsorption capacity of aluminum adjuvant for the relevant antigen (V-01) was further investigated. The stock solution was diluted to 1 mg / mL using a stock solution buffer (20 mM acetate-sodium acetate, 44.2 mg / mL trehalose dihydrate, 0.2 mg / mL polysorbate-80). Aluminum hydroxide adjuvant was diluted to 2, 1, 0.4, 0.2, 0.1, 0.04, 0.02, 0.01, 0.004, and 0.002 mg / mL using buffer 2 (20 mM acetate-sodium acetate, 44.2 mg / mL trehalose dihydrate, 0.2 mg / mL polysorbate 80, 160 mM sodium chloride). The resulting samples were designated as aluminum adjuvant samples. An equal volume of the 1 mg / mL stock solution and the aluminum adjuvant samples were mixed. After thorough mixing, the samples were centrifuged at 6500 × g for 5 min, and the supernatant was examined for protein content using a UV detector. A standard curve was plotted with aluminum adjuvant concentration on the x-axis and supernatant protein content on the y-axis, showing the adsorption capacity of aluminum hydroxide adjuvant for V-01 protein. Figure 4 Extrapolation yields the aluminum adjuvant content corresponding to zero protein content in the supernatant, which represents the maximum adsorption capacity of the aluminum adjuvant.

[0185] Depend on Figure 4 visible, Figure 4The adsorption exhibits good linearity. When the extrapolated supernatant protein content is zero (y = 0), the aluminum content is 0.28 mg / mL. Using this aluminum content as the denominator and the protein content of 0.5 mg / mL as the numerator, the maximum adsorption capacity is calculated as 0.5 mg / mL V-01 protein / 0.28 mg / mL aluminum adjuvant = 1.8 mg V-01 protein / mg aluminum adjuvant. That is, with an aluminum content of 0.5 mg / mL in the finished product, it can adsorb a maximum of 0.9 mg / mL V-01 protein. In other words, the maximum permissible V-01 protein content in the finished product is 0.9 mg / mL.

[0186] Example 7: Stability of finished products with different protein concentrations

[0187] To verify the stability of the finished recombinant fusion protein vaccine formulation at different protein concentrations.

[0188] Experimental groups of finished products with different specifications were set up: the V-01 recombinant fusion protein content in the recombinant fusion protein vaccine formulation was 0.01, 0.02, 0.05, and 0.1 mg / mL, respectively. Only the protein concentration differed among the groups; the composition and content of excipients were the same. Accelerated stability data of the finished products were analyzed, and the results are shown in Table 13. After accelerated stability testing at 25±2℃ for 3 or 6 months, all stability indicators (general physicochemical properties, adsorption rate, biological activity, etc.) showed no significant changes and remained within the quality standard range. This indicates that the finished products with different protein concentrations are all stable.

[0189]

[0190] Example 8: Validation of Prescription Robustness

[0191] To demonstrate that the formulation can be stably stored within a certain range, its robustness is verified. Formulation robustness includes three parts: First, formulation determination. Second, robustness of the bulk formulation. Third, robustness of the finished product formulation.

[0192] 1. Prescription confirmation

[0193] The final formulation of V-01 stock solution was determined to be: 2 mg / mL V-01 recombinant fusion protein, 20 mM acetate-sodium acetate, 44.2 mg / mL trehalose, 0.20 mg / mL polysorbate-80, pH 4.6.

[0194] The recombinant fusion protein V-01 vaccine formulation is available in multiple specifications. The formulation consists of: 0.02-0.10 mg / mL V-01 recombinant fusion protein, 0.5 mg / mL aluminum hydroxide adjuvant, 20 mM acetate-sodium acetate, 44.2 mg / mL trehalose, 4.68 mg / mL sodium chloride, 0.20 mg / mL polysorbate 80, and pH 4.6.

[0195] 2. Robustness verification of the original formulation

[0196] According to the quality standard range requirements, the acceptable pH range is 4.3–4.9; the acceptable protein content range is 1.4–2.6 mg / mL; the acceptable polysorbate-80 content range is 0.1–0.3 mg / mL; the study range for acetate buffer pair content is 18–22 mM; and the study range for trehalose content is 39.78–48.62 mg / mL. Five formulations were designed according to the lower limit, middle, and upper limit of the range. The robustness design of the stock solution formulation is detailed in Table 14.

[0197] The stability under strong acceleration at 30℃, strong acceleration at 25℃, oscillation stability, and freeze-thaw stability of each experimental group were tested. The experimental results are shown in Tables 15-18.

[0198] Table 14 Robust Design of the Original Solution Formulation

[0199] Prescription number* pH Protein content (mg / mL) Acetate (mM) Trehalose (mg / mL) Polysorbate-80 (mg / mL) F9 4.3 1.4 18 39.78 0.1 F2 4.6 2.0 20 44.2 0.2 F3 4.6 2.6 22 48.62 0.3 F4 4.9 2.0 20 44.2 0.2 F5 4.9 2.6 22 48.62 0.3

[0200] Note: *F1, F6, F7, and F8 are control samples used in the experiment to confirm that the experimental procedures have no effect on the final results and are unrelated to the robustness of the prescription, so they will not be discussed further.

[0201] Results of accelerated testing at 30℃ for 1 month and at 25℃ for 3 months showed no significant changes in appearance, pH, osmotic pressure, and protein content for the five formulations; purity showed a slight decreasing trend, but remained within the preset quality standard range; biological activity was within the expected quality standard range. Oscillation stability results showed no significant differences in various test indicators (appearance, pH, osmotic pressure, protein content, purity, and biological activity) between the experimental group (oscillated at 250 rpm for 24 hours at room temperature) and the static control group. Freeze-thaw stability results showed no significant differences in various physicochemical indicators (appearance, pH, osmotic pressure, protein content, and purity) between the experimental group (frozen at -80℃, thawed at 25℃ for 5 cycles) and the zero-temperature group. In summary, the stock solution formulations with 1.4-2.6 mg / mL V-01 protein, 18-22 mM acetate-sodium acetate, 39-49 mg / mL trehalose, 0.1-0.3 mg / mL polysorbate 80, and pH 4.3-4.9 exhibit good robustness.

[0202]

[0203]

[0204]

[0205]

[0206] 3. Robustness verification of formulation

[0207] To demonstrate that the finished formulation can be stably stored under certain conditions, its robustness is verified.

[0208] Since the commercially available product has a protein concentration of 0.02 mg / mL V-01 protein, a 10 μg (0.5 mL) / vial specification was selected for a robustness study of the finished product formulation. The factors influencing the robustness of the finished product formulation were pH (4.3-4.9), aluminum content (0.4-0.6 mg / mL), and sodium chloride content (70-90 mM). Three formulations were designed according to the lower limit, middle, and upper limit of the range, as shown in the table. The stock solution was prepared according to the formulation, and the intermediate was obtained after dilution. The intermediate was then mixed with aluminum adjuvant at a 3:1 volume ratio for adsorption to obtain the finished product. Accelerated stability studies were conducted at 40℃, accelerated stability studies at 25℃, and oscillation stability studies. The data are shown in Tables 20-22.

[0209] Tables 20-22 show that accelerated stability data indicates no significant difference in various test indicators (appearance, pH, osmotic pressure, aluminum content, and adsorption rate) after one month at 40℃ compared to the initial value. Accelerated stability data also indicates no significant difference in various test indicators after three months at 25℃ compared to the initial value. Oscillating stability data shows no significant difference in various test indicators (appearance, pH, osmotic pressure, aluminum content, adsorption rate, and biological activity) between the experimental group (oscillation at 250 rpm for 48 hours at room temperature) and the control group (stationary at room temperature for 48 hours). In summary, the finished product formulation containing 0.4-0.6 mg / mL aluminum hydroxide adjuvant, 70-90 mM sodium chloride, and a pH of 4.3-4.9 exhibits good robustness.

[0210]

[0211]

[0212] The embodiments described above are merely illustrative of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the protection scope of this disclosure. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A recombinant novel coronavirus fusion protein vaccine, comprising: a recombinant fusion protein, a buffer solution, a stabilizer, a surfactant, sodium chloride, and an adjuvant, wherein, The recombinant fusion protein comprises interferon or a functional fragment thereof, SARS-CoV-2 or a functional fragment thereof, and IgG1 Fc; The concentration of the recombinant fusion protein is 0.01-0.1 mg / mL; The stabilizer is trehalose, and the trehalose has a mass-volume percentage of 4%-6%. The concentration of sodium chloride is 70-90 mM; The adjuvant is aluminum hydroxide, and the concentration of aluminum hydroxide is 0.4-0.6 mg / mL; The pH of the vaccine is 4.3-4.9; The amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO 1; The buffer solution is an acetate buffer solution; The surfactant is polysorbate.

2. The vaccine according to claim 1, wherein, The functional fragment of SARS-CoV-2 is its receptor-binding domain (RBD).

3. The vaccine according to claim 1, wherein, The concentration of the buffer solution is 5-80 mM.

4. The vaccine according to claim 3, wherein, The concentration of the buffer solution is 18-22 mM.

5. The vaccine according to claim 1, wherein, The buffer solution is an acetate-sodium acetate buffer solution.

6. The vaccine according to claim 1, wherein, The polysorbate is a polyoxyethylene dehydrated sorbitol fatty acid ester.

7. The vaccine according to claim 6, wherein, The polysorbate is polysorbate 20 or polysorbate 80.

8. The vaccine according to claim 1, wherein, The concentration of the surfactant is 0.01-2 mg / mL.

9. The vaccine according to claim 8, wherein, The concentration of the surfactant is 0.1-0.3 mg / mL.

10. Use of the vaccine according to any one of claims 1-9 in the preparation of a medicament for the prevention and / or treatment of novel coronavirus SARS-CoV-2 infection and / or novel coronavirus disease COVID-19.

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