Fusion protein vaccine and application thereof

Abstract

The invention provides a fusion protein vaccine and application thereof. Specifically, the invention provides a fusion protein and a pharmaceutical composition of the fusion protein and an STING agonist, and provides application of the pharmaceutical composition. According to the fusion protein vaccine disclosed by the invention, the titers of a specific neutralizing antibody and a binding antibody are remarkably improved, the broad spectrum and durability are enhanced, and the immune response of T cells is remarkably improved; besides, the fusion protein vaccine promotes the generation of memory T and B cells, effectively improves immune memory induced by the vaccine, provides a new strategy for preventing and / or treating diseases induced by respiratory tract infection, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, it relates to a fusion protein vaccine and its application. Background Technology

[0002] While current vaccines against respiratory viruses such as COVID-19 and influenza can provide effective immune protection in the short term, they generally face the dual challenges of rapid decline in protective antibodies and insufficient maintenance of cellular immune memory. For example, the neutralizing antibodies produced by most COVID-19 vaccines drop below the required level of protection within a relatively short period of time, and the protection rate of almost all COVID-19 vaccines is below 50% six months after vaccination.

[0003] Furthermore, the bone marrow plasma cells (BMPCs) induced by influenza vaccines have a short lifespan. Studies have shown that the number of specific BMPCs increases four weeks after immunization with seasonal influenza vaccines, but usually returns to pre-vaccination levels relatively quickly.

[0004] Therefore, there is an urgent need in this field to develop new, highly effective, safe vaccines that can simultaneously induce broad-spectrum and durable immune protection against respiratory viruses. Summary of the Invention

[0005] The purpose of this invention is to provide a fusion protein vaccine that induces broad-spectrum and potent immune memory against respiratory viruses and its application.

[0006] Specifically, the purpose of this invention is to provide a fusion protein of an OX40 ligand (OX40L) and a SARS-CoV-2 receptor-binding domain (RBD), and its combination and application with a STING agonist.

[0007] In a first aspect of the invention, a fusion protein is provided, the fusion protein comprising the following elements fused together:

[0008] (a) Respiratory pathogen antigenic elements;

[0009] (b) Fc fragment; and

[0010] (c) OX40L or its active fragment.

[0011] In another preferred embodiment, the pathogen includes: a virus, bacteria, or a combination thereof.

[0012] In another preferred embodiment, the pathogen antigen is an antigen derived from a protein selected from the group consisting of viral envelope proteins, capsid proteins, or combinations thereof.

[0013] In another preferred embodiment, the pathogen antigen is an antigen derived from a protein selected from the group consisting of bacterial surface proteins, capsular polysaccharides, or combinations thereof.

[0014] In another preferred embodiment, the pathogen antigen is a protein or fragment selected from the group consisting of: the S protein (spike protein) and / or N protein (nucleocapsid) of SARS-CoV virus; the F protein (fusion protein) and / or G protein (adhesion glycoprotein) of RSV (respiratory syncytial virus); the HA (hemagglutinin), NA (neuraminidase), NP (nucleoprotein) and / or M1 protein (matrix protein) of influenza virus (A / B / C); the VP1 protein of rhinovirus (RV); or a combination thereof.

[0015] In another preferred embodiment, the pathogen antigen is the S protein (spike protein) of the SARS-CoV-2 virus or an antigenic fragment thereof (such as the RBD fragment).

[0016] In another preferred embodiment, element (b) Fc fragment is located in the middle of the fusion protein, while elements (a) and (c) are located at the ends of the fusion protein, respectively.

[0017] In another preferred embodiment, the elements (a), (b), and (c) are fused sequentially from the N-terminus to the C-terminus in the fusion protein.

[0018] In another preferred embodiment, the fusion protein has the structure shown in Formula I:

[0019] Z1-Z2-L-Z3(I)

[0020] In the formula,

[0021] Z1 is a respiratory pathogen antigen element (preferably, a receptor domain RBD fragment element of the respiratory virus SARS-CoV-2).

[0022] Z2 is the Fc fragment of IgG.

[0023] L represents a linker peptide element or is absent; and

[0024] Z3 is an OX40L component.

[0025] The hyphens "-" represent peptide bonds or linking peptides independently.

[0026] In another preferred embodiment, the RBD fragment has an amino acid sequence as shown in SEQ ID NO:1.

[0027] In another preferred embodiment, the RBD fragment is derived from a variant of the respiratory virus SARS-CoV-2.

[0028] In another preferred embodiment, the SARS-CoV-2 variant is selected from the group consisting of Delta variant, Alpha variant, Beta variant, Gamma variant, Omicron variant, Lambda variant, Mu variant, and Epsilon variant; preferably, it is an Omicron variant or a Delta variant.

[0029] In another preferred embodiment, the RBD fragment contains amino acids 329-528 of the S1 protein of the respiratory virus SARS-CoV-2.

[0030] In another preferred embodiment, the RBD fragment loses its biological activity but retains its immunogenicity.

[0031] In another preferred embodiment, the Fc fragment of the IgG comprises the Fc fragment of human IgG.

[0032] In another preferred embodiment, the Fc fragment of the IgG is selected from the group consisting of the Fc fragments of IgG1, IgG2, IgG3, IgG4, or combinations thereof.

[0033] In another preferred embodiment, the Fc fragment of the IgG is IgG1.

[0034] In another preferred embodiment, the Fc fragment of the IgG has an amino acid sequence as shown in SEQ ID NO:2.

[0035] In another preferred embodiment, the linker peptide is a linker sequence.

[0036] In another preferred embodiment, the length of the peptide linker is 0-300 amino acids, more preferably 1-100 amino acids, and even more preferably 1-30 amino acids.

[0037] In another preferred embodiment, the linker peptide is selected from the following sequence: (G a S b ) x —(G m S n ) y , where a, b, m, n, x, y = 0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a = 4 and b = 1, m = 3 and n = 1).

[0038] In another preferred embodiment, the sequence of the linker peptide is (G4S)4.

[0039] In another preferred embodiment, the linker peptide has an amino acid sequence as shown in SEQ ID NO:3.

[0040] In another preferred embodiment, the OX40L element has an amino acid sequence as shown in SEQ ID NO:4.

[0041] In another preferred embodiment, the OX40L comprises amino acids 51-198 of OX40L.

[0042] In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO:5.

[0043] In another preferred embodiment, the amino acid sequence of the fusion protein has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:5; more preferably, at least 95% sequence identity; and even more preferably at least 96%, 97%, 98%, or 99% sequence identity.

[0044] In another preferred embodiment, the fusion protein is selected from the group consisting of monomers, dimers, and preferably dimers.

[0045] In another preferred embodiment, the fusion protein has the following characteristics:

[0046] (1) Significantly enhanced immunogenicity; and / or

[0047] (2) Z1, Z2 and Z3 do not affect each other in terms of structure, which ensures that the RBD is correct in spatial structure.

[0048] In a second aspect of the invention, a combination of active ingredients is provided, the components of which include: (a) the fusion protein or its coding sequence or its expression vector as described in the first aspect of the invention and (b) an adjuvant, said adjuvant being a STING agonist.

[0049] In another preferred embodiment, the components of the combination consist of: (a) the fusion protein and (b) the STING agonist.

[0050] In another preferred embodiment, the STING agonist (or a pharmaceutically acceptable salt thereof) is selected from the group consisting of cyclic dinucleotide agonists (CDNs, such as cGAMP), non-nucleotide small molecule agonists (such as MSA-2, CF501), metal ion agonists (such as manganese ion salts), polymeric agonists (such as chitosan), or nano-adjuvants, or combinations thereof; preferably non-nucleotide small molecule agonists (such as CF501) or cyclic dinucleotide agonists (CDNs, such as cGAMP).

[0051] In a third aspect of the invention, a pharmaceutical composition is provided, comprising:

[0052] (a) A fusion protein as described in the first aspect of the present invention, or its coding sequence or expression vector thereof, as an active ingredient;

[0053] (b) Pharmaceutically acceptable carriers.

[0054] In another preferred embodiment, the pharmaceutical composition is a vaccine composition.

[0055] In another preferred embodiment, the pharmaceutical composition further comprises (c) a first adjuvant, which is a STING agonist or a pharmaceutically acceptable salt thereof.

[0056] In another preferred embodiment, the pharmaceutical composition further comprises (d) an additional second adjuvant, which is different from the first adjuvant.

[0057] In another preferred embodiment, the STING agonist or a pharmaceutically acceptable salt thereof activates or indirectly activates the OX40L element in the fusion protein.

[0058] In another preferred embodiment, the STING agonist (or a pharmaceutically acceptable salt thereof) is selected from the group consisting of cyclic dinucleotide agonists (CDNs, such as cGAMP), non-nucleotide small molecule agonists (such as MSA-2, CF501), metal ion agonists (such as manganese ion salts), polymeric agonists (such as chitosan), or nano-adjuvants, or combinations thereof; preferably non-nucleotide small molecule agonists (such as CF501) or cyclic dinucleotide agonists (CDNs, such as cGAMP).

[0059] In another preferred embodiment, the pharmaceutically acceptable carrier contains a liquid, preferably water, saline, or a buffer solution.

[0060] In another preferred embodiment, the carrier further contains auxiliary substances, preferably fillers, lubricants, flow aids, wetting agents or emulsifiers, pH buffers, etc.

[0061] In a fourth aspect of the invention, a polynucleotide is provided that encodes a fusion protein as described in the first aspect of the invention.

[0062] In another preferred embodiment, the polynucleotide is selected from the group consisting of DNA sequences, RNA sequences, or combinations thereof; preferably DNA.

[0063] In another preferred embodiment, the polynucleotide further comprises, flanking the ORF of the fusion protein, an auxiliary element selected from the group consisting of: signal peptides, secretory peptides, tag sequences (such as 6His), or combinations thereof.

[0064] In a fifth aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the fourth aspect of the invention.

[0065] In another preferred embodiment, the vector is used to express the fusion protein.

[0066] In another preferred embodiment, the vector is selected from the group consisting of plasmid vectors, viral vectors, or combinations thereof; preferably, it is a plasmid vector.

[0067] In a sixth aspect of the invention, a host cell is provided, wherein the host cell contains the vector described in the fifth aspect of the invention or the genome of which the polynucleotides described in the fourth aspect of the invention are integrated.

[0068] In another preferred embodiment, the cells are selected from the group consisting of isolated cells, genetically engineered cells, or combinations thereof.

[0069] In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0070] In another preferred embodiment, the host cell is selected from the group consisting of: Escherichia coli, insect cells, SF9, HeLa, HEK293 (such as EXPi293T cells), CHO, yeast cells, or combinations thereof.

[0071] In another preferred embodiment, the host cell is selected from the group consisting of BL21 (DE3), Rosetta (DE3), and OrigamiB (DE3).

[0072] In another preferred embodiment, the host cell is a Chinese hamster ovary cell (CHO).

[0073] In a seventh aspect of the invention, there is provided the use of a combination of active ingredients as described in the second aspect of the invention for preparing a vaccine composition or vaccine for the prevention and / or treatment of diseases induced by respiratory tract infections, wherein,

[0074] The vaccine composition or vaccine comprises: (i) a fusion protein as described in the first aspect of the invention, or its coding sequence or expression vector thereof, as an active ingredient; and (ii) the STING agonist or a pharmaceutically acceptable salt thereof.

[0075] In another preferred embodiment, the respiratory infection-induced disease is selected from the group consisting of: respiratory viral infections, respiratory bacterial infections, or combinations thereof; preferably, it is a respiratory viral infection-induced disease.

[0076] In another preferred embodiment, the vaccine composition or vaccine further comprises: (iii) an additional adjuvant.

[0077] In another preferred embodiment, the vaccine composition or vaccine is preventative and / or therapeutic.

[0078] In another preferred embodiment, the vaccine composition or vaccine is a unit dosage form.

[0079] In another preferred embodiment, the vaccine composition or vaccine is monovalent or multivalent.

[0080] In another preferred embodiment, the adjuvant is selected from the group consisting of aluminum salts, water-in-oil emulsions, oil-in-water emulsions, nanoparticles, microparticles, liposomes, immunostimulatory complexes, or combinations thereof.

[0081] In another preferred embodiment, the adjuvant is selected from the group consisting of: muramyl dipeptide and its derivatives, saponins, lipid A, cytokines, derived polysaccharides, bacterial toxins, microorganisms and their products such as mycobacteria (tuberculosis bacillus, BCG), cloacae, Bordetella pertussis, propolis, or combinations thereof.

[0082] In another preferred embodiment, the amount of the fusion protein in the unit dosage form vaccine composition or vaccine is 1-100 μg, more preferably 2-50 μg, and even more preferably 5-25 μg.

[0083] In another preferred embodiment, the concentration of the STING agonist or a pharmaceutically acceptable salt thereof in the vaccine composition or vaccine is 0.01 μg / μL to 100 μg / μL, more preferably 0.1 μg / μL to 20 μg / μL, and even more preferably 0.15 μg / μL to 5 μg / μL.

[0084] In another preferred embodiment, the ratio of component (a) to component (b) in a unit dosage form of the vaccine composition satisfies:

[0085] 1-20 μg fusion protein; 2-100 μg STING agonist or a pharmaceutically acceptable salt thereof.

[0086] In another preferred embodiment, the ratio of component (a) to component (b) in a unit dosage form of the vaccine composition satisfies:

[0087] 5±2μg fusion protein: 20±5μg STING agonist or a pharmaceutically acceptable salt thereof.

[0088] In another preferred embodiment, the vaccine composition is in the form of an injection (such as an intramuscular injection) or a mucosal immunomodulator (such as nasal drops).

[0089] In another preferred embodiment, a therapeutically effective amount of component (b) is administered to the subject in need before, during, and / or after the administration of component (a) of the vaccine composition.

[0090] In another preferred embodiment, the subject is a patient (or subject) diagnosed with a disease induced by a respiratory viral infection.

[0091] In another preferred embodiment, the subject is a human or non-human mammal, preferably a rodent (such as a mouse or rat) or a primate (such as a human or monkey).

[0092] In another preferred embodiment, the vaccine is administered to a human or a non-human mammal.

[0093] In another preferred embodiment, the vaccine is administered to a subject diagnosed with a respiratory viral infection or a subject who is susceptible (or at risk).

[0094] In another preferred embodiment, the disease induced by the respiratory virus infection is selected from the group consisting of: cold, influenza, viral pneumonia, viral bronchitis and / or bronchiolitis, viral pharyngitis, herpetic pharyngitis, pharyngoconjunctival fever, or a combination thereof; preferably viral pneumonia, and more preferably novel coronavirus infection.

[0095] In another preferred embodiment, the vaccine has features selected from the group consisting of:

[0096] (1) Significantly increased levels of specific neutralizing antibodies and / or binding antibodies;

[0097] (2) Prolong the duration of neutralizing and binding antibodies;

[0098] (3) Significantly enhanced T-cell immune response;

[0099] (4) Promotes the proliferation of T cells (including Tfh).

[0100] (5) Maintain T cell survival; and / or

[0101] (6) Significantly enhanced proliferative capacity of memory B cells and / or memory T cells;

[0102] (7) Significantly enhanced long-term immune protection.

[0103] In another preferred embodiment, the administration includes intramuscular injection, tail injection, subcutaneous injection, or a combination thereof.

[0104] In another preferred embodiment, the object includes an animal model.

[0105] In another preferred embodiment, the object includes a human or a non-human mammal, preferably a human.

[0106] In another preferred embodiment, the drug or formulation is administered to the subject, thereby imparting to the subject a significantly enhanced level of antigen-specific antibodies and / or immune memory.

[0107] In another preferred embodiment, the drug or preparation further includes: other drugs for treating diseases induced by respiratory viral infections.

[0108] In another preferred embodiment, the other drugs for treating diseases induced by respiratory viral infections are selected from the group consisting of small molecule compounds, antibody drugs, antibody-drug conjugates (ADCs), or combinations thereof.

[0109] In another preferred embodiment, the drug or formulation may also be administered in combination with a therapy.

[0110] In another preferred embodiment, the therapy is selected from the group consisting of surgical treatment, drug treatment, or a combination thereof.

[0111] In an eighth aspect of the invention, a method for preparing the fusion protein is provided, comprising the following steps:

[0112] (i) Under suitable expression conditions, host cells as described in the sixth aspect of the present invention are cultured to obtain a culture medium containing the expressed protein;

[0113] (ii) The fusion protein is isolated from the culture medium to obtain the isolated fusion protein.

[0114] In another preferred embodiment, the method further includes the step of:

[0115] (iii) The obtained fusion protein is purified.

[0116] In another preferred embodiment, the expressed fusion protein is expressed in the host cell as a fusion protein inclusion body.

[0117] In another preferred embodiment, the method for preparing the fusion protein includes the following steps:

[0118] (1) Culture the host cells (e.g., EXPi293T) for 2-5 days until the cell density reaches 4×10⁻⁶. 6 / mL-5×10 6 After passing the sample at a concentration of 1 mL, subculture and transfection are performed, preferably at a concentration of 2 × 10⁶. 6 / mL-3×10 6 / mL (e.g., 2.5×10) 6 / mL);

[0119] (2) Using the vector as described in the fifth aspect of the present invention, transfect the cells obtained in step (1) to construct a cell line expressing the fusion protein.

[0120] (3) Continue culturing for 5-15 days (e.g., 7 days);

[0121] (4) Collect the cell culture medium from step (3) and purify it using affinity chromatography.

[0122] In a ninth aspect of the invention, a kit is provided, the kit comprising:

[0123] (A) A first container, and a fusion protein as described in the first aspect of the invention, a polynucleotide as described in the fourth aspect of the invention, or a vector as described in the fifth aspect of the invention, located within the first container; and

[0124] (B) A second container, and a STING agonist or a pharmaceutically acceptable salt thereof located in the second container.

[0125] In another preferred embodiment, the first container and the second container are different containers.

[0126] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0127] Figure 1 The effects of STING agonists on OX40 expression in T cells were shown: (A) The STING agonist CF501 acted on spleen cells in vitro, affecting their CD3 expression. + (B) Effects of intramuscular injection of the STING agonist CF501 on CD3 expression in mouse inguinal lymph nodes. + Influence on the expression of OX40 protein on the surface of T cells.

[0128] Figure 2 The diagram shows a schematic of the connection of the fusion protein of the present invention (top) and a schematic of the expression plasmid map of pFUSE-hIgG1-Fc2 (containing the OX40L protein sequence) (bottom).

[0129] Figure 3 The gel electrophoresis test results of the fusion protein of the present invention are shown.

[0130] Figure 4 The effects of the fusion protein of the present invention on gene expression in mouse lymph node tissues were demonstrated.

[0131] Figure 5 The effect of the fusion protein of the present invention on gene expression in T cells in vitro in spleen cells was demonstrated.

[0132] Figure 6 The muscle and mucosal immune processes in mice were shown.

[0133] Figure 7 The antibody titer of SARS-CoV-2 (Delta variant) RBD-specific IgG in mouse serum was shown after two immunizations (i.e., on day 35 of immunization).

[0134] Figure 8 The study showed that, three times after immunization (i.e., on day 63 of immunization), the SARS-CoV-2 (Delta variant) 2RBD-specific IgG in mouse serum was [value missing]. Figure 8 A) IgG1 ( Figure 8 B) IgG2a Figure 8 C) antibody titer.

[0135] Figure 9 The results show the titer of neutralizing antibodies against SARS-CoV-2 (Delta variant) pseudovirus in mouse serum after two immunizations (i.e., on day 35 of immunization).

[0136] Figure 10 The results showed that, after three immunizations of mice (i.e., on day 63 of immunization), the Delta variant of SARS-CoV-2 was detected in mouse serum. Figure 10 A), BA.2.2 variant ( Figure 10 B), BA.5 Figure 10 C) Neutralizing antibody titers of pseudoviruses in mutant strains.

[0137] Figure 11 The titer of neutralizing antibodies against SARS-CoV pseudovirus in mouse serum was shown after three immunizations (i.e., on day 63 of immunization).

[0138] Figure 12 This shows the presence of Tfh cells in mouse lymph nodes after three immunizations (i.e., on day 63 of immunization). Figure 12 A) GC B cells ( Figure 12 B) and memory B cells (i.e., RBD-specific MB cells) Figure 12 C) Flow cytometry analysis.

[0139] Figure 13 The figures show the results on day 30 after the third immunization (i.e., one month after immunization). Figure 13 A) and day 180 (i.e., six months after immunization), Figure 13 B), antibody titer of SARS-CoV-2 (Delta variant) RBD-specific IgG in mouse serum.

[0140] Figure 14 The figures show the results on day 30 after the third immunization (i.e., one month after immunization). Figure 14 A) and day 180 (i.e., six months after immunization), Figure 14 B), the titer of neutralizing antibodies against SARS-CoV-2 (Delta variant) pseudovirus in mouse serum.

[0141] Figure 15This shows that after two mucosal immunizations of mice (i.e., on day 35 of immunization), the level of SARS-CoV-2 (Delta variant) RBD-specific IgG in mouse serum was [data missing]. Figure 15 A) and IgA( Figure 15 B) antibody titer.

[0142] Figure 16 This shows the presence of SARS-CoV-2 (Delta variant) RBD-specific IgG in mouse serum after three immunizations via mucosal membrane (i.e., on day 63 of immunization). Figure 16 A) and IgA( Figure 16 B) antibody titer.

[0143] Figure 17 The results showed that, after two separate immunizations of mice via the mucosa (i.e., on day 35 after immunization), Figure 17 A) and after three immunizations of mice via the mucosa (i.e., on day 63 of immunization), Figure 17 B), the titer of neutralizing antibodies against SARS-CoV-2 (Delta variant) pseudovirus in mouse serum. Detailed Implementation

[0144] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that activating the STING signaling pathway can effectively upregulate OX40 on the surface of T cells, further stimulating the production of high antibody titers in animals and significantly improving immunogenicity. Based on this discovery, the inventors have provided a vaccine combination using a subunit vaccine against SARS-CoV-2 as an example. Specifically, the inventors tandemly synthesized a fusion protein by linking the receptor-binding domain (RBD), IgG1 Fc, and OX40L protein of the novel coronavirus (SARS-CoV-2) with a connecting arm. These components do not interfere with each other structurally, ensuring the correct spatial structure of the RBD while increasing immunogenicity.

[0145] Based on this, the inventors combined the fusion protein of this invention with the STING agonist and found that the combined use of the two synergistically induces immune memory, thereby developing a fusion protein vaccine against respiratory viruses. This vaccine significantly increases the levels of specific neutralizing and binding antibodies and significantly prolongs the duration of neutralizing and binding antibodies, exhibiting strong immunogenicity and a high level of immune memory. Based on this, the present invention was completed.

[0146] the term

[0147] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0148] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0149] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0150] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0151] As used herein, the term "autologous" refers to something derived from the same organism. For example, an autologous sample (e.g., cells) can refer to a sample that is removed, processed, and then returned to the same subject (e.g., a patient) at a later time. In terms of process, autologous homologous processes can be distinguished from allogeneic processes, in which the donor and recipient of the sample (e.g., cells) are not the same subject.

[0152] As used herein, the term "alien" refers to any material derived from a different animal of the same species as the individual into which the material was introduced. Two or more individuals are said to be alliens of each other when the genes at one or more loci are not identical. In some respects, allien materials from individuals of the same species may be genetically completely different, thus exhibiting antigenic interactions.

[0153] As used in this article, the term "heterogeneous" refers to a graft derived from an animal of a different species.

[0154] The terms “give” or “administer” refer to the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0155] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to vertebrates, preferably mammals such as humans. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sporting animals, and pets. This also includes tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0156] As used herein, the terms “treatment” and “treatment” refer to methods for obtaining a beneficial or desired outcome, including but not limited to therapeutic and / or preventative benefits. For example, treatment may include the administration of the systems or cell populations disclosed herein. A therapeutic benefit is any treatment-related improvement or effect on one or more conditions (e.g., a disease or symptom) during treatment. For preventative benefits, the composition may be administered to subjects at risk of developing a specific condition, or to subjects reporting one or more physiological symptoms of a disease, even if the condition may not yet be present.

[0157] antigenic peptide

[0158] As used herein, the terms "antigenic peptide," "pathogen antigen," and "pathogen antigenic peptide" are used interchangeably and refer to a peptide from another protein intended to induce an immune response in an animal. This antigenic peptide is relative to the carrier protein and does not refer to a peptide segment of the carrier protein itself that can elicit an immune response. Typically, an antigenic peptide refers to the peptide segment that the immune response intends to target, rather than originating from the carrier protein.

[0159] In this invention, "antigen peptide" refers to the receptor domain (RBD) of a virus and is a general term for active polypeptides that can bind to receptors on the surface of host cells.

[0160] The present invention provides an antigenic peptide derived from the receptor-binding domain (RBD) of the novel coronavirus (SARS-CoV-2). In a preferred embodiment of the present invention, the antigenic peptide has amino acids 329-528 of the S1 protein of SARS-CoV-2. Preferably, the antigenic peptide according to the present invention has the amino acid sequence shown in SEQ ID NO:1.

[0161] carrier proteins

[0162] As used herein, the term "carrier protein" refers to a protein that serves as the protein structural backbone in the fusion protein of this invention. Typically, the carrier protein is a protein with strong immunogenicity, such as a pathogen protein, and representative examples include (but are not limited to): the Fc fragment of IgG, viral proteins, bacterial proteins, chlamydia proteins, mycoplasma proteins, etc.

[0163] In this invention, the carrier protein is the Fc fragment of IgG.

[0164] Specifically, the Fc fragment of the IgG includes the Fc fragment of human IgG.

[0165] Specifically, the Fc fragment of the IgG is selected from the group consisting of the Fc fragments of IgG1, IgG2, IgG3, and IgG4, or combinations thereof; preferably IgG1.

[0166] Preferably, the Fc fragment of the IgG has the amino acid sequence shown in SEQ ID NO:2.

[0167] RBD

[0168] As used in this article, the terms "RBD", "receptor domain", and "RBD fragment" are used interchangeably. The RBD refers to the S1 subunit of the spike protein (S protein) on the surface of the coronavirus. It is a relatively independent domain that is responsible for binding to receptors on the surface of host cells, thereby mediating viral entry into cells and causing infection. It is the most critical antigenic epitope on the viral surface and can induce the body to produce neutralizing antibodies. It is the core target for vaccine and antibody drug development.

[0169] In this invention, the receptor-binding domain (RBD) of the novel coronavirus (SARS-CoV-2) is used as a key element of the fusion protein of this invention.

[0170] Specifically, the RBD fragment of the present invention has an amino acid sequence as shown in SEQ ID NO:1.

[0171] Preferably, the RBD fragment of the present invention further comprises an RBD-derived protein having RBD function, formed by substituting, deleting or adding one or more (usually 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid residues of the amino acid sequence shown in SEQ ID NO:1.

[0172] Preferably, the amino acid sequence of the RBD fragment of the present invention further comprises a protein or its active fragment that has ≥80% homology, more preferably ≥85%, more preferably ≥90%, and most preferably ≥95% homology to the amino acid sequence defined by the RBD and has RBD function; or

[0173] Preferably, the RBD segment of the present invention further includes a segment with RBD functionality.

[0174] OX40L and OX40

[0175] As used in this article, the term "OX40" refers to a member of the tumor necrosis factor receptor (TNFR) family, also known as CD134 or TNFRSF4, which is mainly expressed on the surface of activated CD4+ and CD8+ T cells, while its ligand OX40L (CD252) is enriched in antigen-presenting cells (such as dendritic cells).

[0176] As used in this article, the term "OX40L" refers to the ligand of OX40, which belongs to the tumor necrosis factor superfamily (TNFSF4) and plays a key role in immune response processes such as T cell activation and proliferation, memory T cell formation, and regulation of immune homeostasis.

[0177] As used in this article, the terms “OX40-OX40L” and “OX40-OX40L pathway” are used interchangeably. They refer to a T cell co-stimulatory signaling pathway that plays an important role in inducing immune memory. It mainly enhances the production of memory T cells by promoting T cell proliferation, differentiation and maintaining T cell survival (The Journal of Experimental Medicine vol. 191, 2(2000): 365-74.).

[0178] Specifically, OX40 can significantly promote T cell clonal expansion, enhance T cell survival, and maintain the long-term survival of effector T cells, thereby promoting their differentiation into memory T cells. Furthermore, activation of the OX40 pathway helps promote the formation of memory B cells and plays an important role in the differentiation of Tfh cells.

[0179] In this invention, OX40L is used as another key element of the fusion protein of this invention.

[0180] Specifically, the OX40L of the present invention has the amino acid sequence shown in SEQ ID NO:4.

[0181] Preferably, the OX40L of the present invention further comprises a protein derived from OX40L that has OX40L function and is formed by substitution, deletion or addition of one or more (usually 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid residues of the amino acid sequence shown in SEQ ID NO:4.

[0182] Preferably, the amino acid sequence of OX40L according to the present invention further comprises a protein or its active fragment having ≥80%, more preferably ≥85%, more preferably ≥90%, and most preferably ≥95% homology with the amino acid sequence defined by OX40L and having OX40L function.

[0183] Preferably, the OX40L segment of the present invention further includes a segment having OX40L functionality.

[0184] Fusion protein

[0185] As used herein, the terms “recombinant fusion protein,” “protein of the present invention,” “fusion protein of the present invention,” “recombinant fusion protein of the present invention,” “fusion protein,” “engineered protein,” “polypeptide of the present invention,” and “active polypeptide of the present invention” are used interchangeably and refer to having the structure described in Formula Ia of the first aspect of the present invention.

[0186] Specifically, the fusion protein includes the following elements fused together:

[0187] (a) Respiratory pathogen antigenic elements;

[0188] (b) Fc fragment; and

[0189] (c) OX40L or its active fragment.

[0190] Preferably, the fusion protein of the present invention is a recombinant fusion protein RBD-Fc-OX40L obtained by fusing the receptor-binding domain (RBD), IgG1 Fc and OX40L protein of the novel coronavirus (SARS-CoV-2).

[0191] Specifically, the structure of the fusion protein is shown as Z1-Z2-L-Z3 (Formula I).

[0192] Wherein, Z1 is a respiratory pathogen antigen element (preferably, a receptor domain RBD fragment element of the respiratory virus SARS-CoV-2); Z2 is an Fc fragment of IgG; L is a linker peptide element or none; and Z3 is an OX40L element; "-" represents a peptide bond or linker peptide independently.

[0193] In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO:5.

[0194] As used herein, the term "fusion protein" also includes variations of fusion proteins (such as the sequence shown in SEQ ID NO:5) having the above-described activities. These variations include (but are not limited to): deletions, insertions, and / or substitutions of 1-3 amino acids (typically 1-2, more preferably 1), and additions or deletions of one or more amino acids (typically up to 3, preferably up to 2, more preferably up to 1) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties typically does not alter the function of the protein. Similarly, additions or deletions of one or more amino acids at the C-terminus and / or N-terminus typically do not alter the structure and function of the protein. Furthermore, the term also includes polypeptides of the invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0195] This invention also includes active fragments, derivatives, and analogs of the aforementioned fusion proteins. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a polypeptide that substantially retains the function or activity of the fusion protein of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) polypeptides having substituent groups in one or more amino acid residues, or (iii) polypeptides formed by fusing an antigenic peptide with another compound (e.g., a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (fusion proteins formed by fusing with a leader sequence, secretion sequence, or tag sequence such as 6×His). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0196] A preferred class of active derivatives refers to polypeptides formed by replacing up to three, more preferably up to two, and even more preferably up to one amino acid with an amino acid of similar or analogous properties compared to the amino acid sequence of Formula I. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0197] Table A

[0198] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0199] The present invention also provides analogs of the fusion protein of the present invention. These analogs may differ from any of the polypeptides shown in SEQ ID NO.:5 in that they may differ in amino acid sequence, in the form of modifications that do not affect the sequence, or both. Analogs also include those having residues different from native L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0200] Modifications (typically without altering the primary structure) include chemically derived forms of peptides, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications performed during peptide synthesis and processing or further processing steps. This modification can be accomplished by exposing the peptide to glycosylating enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Modifications also include peptides modified to improve their resistance to proteolysis or optimize their solubility.

[0201] The polypeptides (fusion proteins) of this invention can also be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases. These salts include (but are not limited to) salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or hydroxyethanesulfonic acid. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium), and salts in the form of esters, carbamates, or other conventional "prodrugs."

[0202] Polynucleotides

[0203] The term "polynucleotide of the present invention" can refer to a polynucleotide that encodes the fusion protein of the present invention, or it can refer to a polynucleotide that also includes additional coding and / or non-coding sequences.

[0204] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the fusion protein of this invention can be identical to the sequence encoding the fusion protein shown in SEQ ID NO:5 or a degenerate variant.

[0205] As used herein, "degenerate variant" refers to a nucleic acid sequence that encodes a fusion protein as shown in SEQ ID NO:5, but with different coding region sequences.

[0206] The present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs, and derivatives of fusion proteins having the same amino acid sequence as those of the present invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the fusion protein of the present invention it encodes.

[0207] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions (or strict conditions). In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%.

[0208] The fusion proteins and polynucleotides of the present invention are preferably provided in isolated form, and more preferably, purified to homogenization.

[0209] As used in this article, "isolated" means that a substance has been separated from its original environment (in the case of a natural substance, the original environment is the natural environment). For example, polynucleotides and fusion proteins in their natural state within living cells are not isolated and purified, but the same polynucleotides or fusion proteins are isolated and purified if they are separated from other substances present in their natural state.

[0210] As used herein, the term "primer" refers to a general term for oligonucleotides that, when paired with a template, can be used by DNA polymerase to synthesize a DNA strand complementary to the template. Primers can be natural RNA, DNA, or any form of natural nucleotide. Primers can even be non-natural nucleotides such as LNA or ZNA. A primer is "probably" (or "substantially") complementary to a specific sequence on one strand of the template. A primer must be fully complementary to one strand of the template to begin elongation, but the primer sequence does not need to be perfectly complementary to the template sequence. For example, adding a non-complementary sequence to the 5' end of a primer that is complementary to the template at the 3' end will still result in a primer that is probably complementary to the template. As long as the primer is long enough to bind sufficiently to the template, even a partially complementary primer can form a primer-template complex with the template, thereby enabling amplification.

[0211] The full-length polynucleotide sequences of the fusion protein or its elements of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the disclosed nucleotide sequences, especially the open reading frame sequences, according to this invention, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0212] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0213] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0214] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0215] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the polynucleotides of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE (RACE-cDNA end amplification) method is preferred. Primers used for PCR can be appropriately selected based on the sequence information disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0216] Expression vectors and host cells

[0217] The present invention also relates to a vector containing a polynucleotide comprising the fusion protein of the present invention, a host cell genetically engineered using the vector or fusion protein encoding sequence of the present invention, and a method for generating the protein of the present invention using recombinant technology.

[0218] Using conventional recombinant DNA techniques, the polynucleotide sequence of this invention can be used to express or produce recombinant proteins. Generally, the following steps are involved:

[0219] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding the fusion protein of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0220] (2) Host cells cultured in a suitable culture medium;

[0221] (3) Isolate and purify proteins from culture media or cells.

[0222] In this invention, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0223] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the protein of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0224] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0225] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0226] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: bacterial cells of Escherichia coli and Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, NSO, COS7, or 293 cells.

[0227] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0228] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0229] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, polyethyleneimine packaging, etc.

[0230] The obtained transformants can be cultured using conventional methods to express the protein or polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0231] The proteins described in the above methods may be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0232] Linking peptides

[0233] As used in this article, the terms "linking peptide" and "peptide linker" are used interchangeably.

[0234] This invention provides a fusion protein that optionally contains a peptide linker. The size and complexity of the peptide linker can affect the activity of the protein. Generally, the peptide linker should have sufficient length and flexibility to ensure that the two linked proteins have sufficient spatial freedom to perform their functions. At the same time, the formation of α-helices or β-sheets in the peptide linker should be avoided to prevent the formation of these structures that could negatively impact the stability of the fusion protein.

[0235] The length of the linker peptide is generally 0-30 amino acids, preferably 0-10 amino acids, and more preferably 0-5 amino acids.

[0236] In another preferred embodiment, the linker peptide has an amino acid sequence as shown in SEQ ID NO:3.

[0237] Diseases caused by respiratory tract infections

[0238] As used in this article, the term "diseases induced by respiratory infections" refers to diseases induced by pathogens such as viruses, bacteria, and mycoplasma.

[0239] Specifically, the respiratory infection-induced disease is selected from the group consisting of: respiratory viral infection-induced disease, respiratory bacterial infection-induced disease, or a combination thereof; preferably, it is a respiratory viral infection-induced disease.

[0240] As used in this article, the terms "diseases induced by respiratory viral infection", "viral respiratory infectious disease", and "respiratory viral infection" are used interchangeably. They refer to infectious diseases caused by pathogenic viruses invading the respiratory tract and are divided into related diseases of viral upper respiratory tract infection and viral lower respiratory tract infection.

[0241] Viral upper respiratory tract infection-related diseases are inflammations of the nasal cavity, pharynx, or larynx caused by viruses, commonly known as "the common cold." The main symptoms include nasal congestion, runny nose, sore throat, cough, and fever. It is the most common infectious disease. Viral upper respiratory tract infection diseases are selected from the following group: common cold, herpetic pharyngitis, viral pharyngitis, pharyngoconjunctival fever, etc.

[0242] Viral lower respiratory tract infection-related diseases are infectious diseases caused by viruses, mainly including acute tracheobronchitis, chronic bronchitis, pneumonia, etc., with clinical symptoms including fever, cough, sputum production, and inflammatory changes on imaging. Viral lower respiratory tract infection diseases are selected from the following group: influenza (flu), viral pneumonia, viral bronchitis, and bronchiolitis, etc.

[0243] Pharmaceutical Compositions and Administration

[0244] (i) Pharmaceutical Composition

[0245] As used herein, the terms “composition,” “composition of the present invention,” “pharmaceutical composition,” and “pharmaceutical composition of the present invention” are used interchangeably and refer to the pharmaceutical composition as described in the second aspect of the present invention.

[0246] When the pharmaceutical compositions of the present invention are used for actual treatment, various dosage forms of the pharmaceutical compositions may be used depending on the application. Preferred dosage forms are injections and nasal drops.

[0247] These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally by adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in the conventional manner depending on the dosage form.

[0248] The pharmaceutical compositions of the present invention can also be administered in a sustained-release form. For example, the active ingredient of the present invention or a salt thereof may be incorporated into a pill or microcapsule carried by a sustained-release polymer, and then the pill or microcapsule may be surgically implanted into human tissue.

[0249] Examples of slow-release polymers include ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, and lactic acid-glycolic acid copolymers. More preferably, biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers are also examples.

[0250] When the pharmaceutical composition of the present invention is used for prevention or treatment, the dosage of the fusion protein of the present invention and the STING agonist or a pharmaceutically acceptable salt thereof as active ingredients may be reasonably determined based on the weight, age, sex, and symptom severity of each subject (patient) to be prevented or treated.

[0251] Specifically, the pharmaceutical composition of the present invention is a vaccine composition.

[0252] The vaccine composition of the present invention can be monovalent (containing only one fusion protein or polynucleotide) or polyvalent (containing multiple fusion proteins or polynucleotides).

[0253] Furthermore, the pharmaceutical compositions of the present invention comprise a combination of the active ingredients of the present invention, said combination of active ingredients including:

[0254] (a) the fusion protein as described in the first aspect of the invention, or its coding sequence or its expression vector, as an active ingredient, and (b) the STING agonist or its pharmaceutically acceptable salt.

[0255] Specifically, the fusion protein of the present invention has excellent specificity and immune activity, so the active ingredient of the present invention can be used to prepare vaccines for immunotherapy or prevention.

[0256] (ii) Vaccine composition

[0257] The present invention provides a vaccine composition (or vaccine) that can be preventive (i.e., preventive of diseases, such as influenza, viral pneumonia, etc., diseases caused by respiratory viral infections) or therapeutic (i.e., treatment of diseases after infection, such as influenza, viral pneumonia, etc., diseases caused by respiratory viral infections).

[0258] These vaccine compositions contain immune antigens (including the fusion proteins of the present invention) or pharmaceutically acceptable salts thereof, and are generally combined with “vaccine-acceptable carriers”, which include any carriers that do not induce antibodies harmful to individuals receiving the composition.

[0259] Suitable carriers are typically large, slowly metabolizing macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), etc. These carriers are well known to those skilled in the art. Additionally, these carriers can act as immunostimulants (“adjuvants”). Furthermore, antigens can also be conjugated to bacterial toxoids (such as toxoids of pathogens like diphtheria, tetanus, cholera, and Helicobacter pylori). Preferably, the vaccine composition (or vaccine) of the present invention is constructed using an RBD fragment of a respiratory virus (such as SARS-CoV-2 infection).

[0260] In this invention, the vaccine composition or vaccine comprises (i) a fusion protein as described in the first aspect of the invention, or its coding sequence or expression vector thereof, as an active ingredient, and (ii) the STING agonist or a pharmaceutically acceptable salt thereof.

[0261] Preferably, the vaccine composition or vaccine further comprises: (iii) an additional adjuvant.

[0262] Specifically, the STING agonist is selected from the group consisting of: cyclic dinucleotides (CDNs, such as cGAMP), non-nucleotide small molecule agonists (such as MSA-2, CF501), metal ion agonists (such as manganese ion salts), polymeric agonists (such as chitosan), or nano-adjuvants, or combinations thereof; preferably non-nucleotide small molecule agonists (such as CF501) or cyclic dinucleotides (CDNs, such as cGAMP).

[0263] Vaccine compositions, including immunogenic compositions (e.g., may include antigens, STING agonists or their pharmaceutically acceptable salts, pharmaceutically acceptable carriers, and adjuvants), typically contain diluents such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc., may be present in these carriers.

[0264] More specifically, vaccines, including immunogenic compositions, contain an immunologically effective amount of an immunogenic fusion protein, as well as the other required components mentioned above. "Immunologically effective amount" refers to the amount administered to an individual as a single or partial dose that is effective for treatment or prevention. This dosage can be determined based on the individual's health and physiological condition, the individual's class (e.g., human), the individual's immune system's ability to synthesize antibodies, the required level of protection, the vaccine formulation, the treating physician's assessment of the medical condition, and other relevant factors. This dosage is expected to be within a relatively wide range and can be determined through routine testing.

[0265] Typically, vaccine compositions or immunogenic compositions can be formulated as injectable preparations, such as liquid solutions or suspensions; they can also be formulated as solid forms suitable for reconstitution into solutions or suspensions or liquid excipients prior to injection. The formulation may also be emulsified or encapsulated in liposomes to enhance adjuvant effects.

[0266] (ii) Application method

[0267] Once formulated into the composition of the present invention, it can be administered directly to the subject. The subject to be treated can be a mammal, particularly a human or a mouse.

[0268] It should be understood that when the subject is a human, the fusion protein of the present invention preferably uses human OX40L protein or its active fragment.

[0269] When used as a vaccine, the fusion protein of the present invention can be administered directly to an individual using known methods, along with a STING agonist or a pharmaceutically acceptable salt thereof. These vaccines are typically administered via the same route of administration as conventional vaccines and / or by mimicking the pathogen infection pathway.

[0270] The routes of administration for the vaccine compositions of the present invention include (but are not limited to): intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, or other parenteral routes. Routes of administration may be combined if necessary, or adjusted according to the disease condition. The vaccine compositions may be administered in single or multiple doses, and may include booster doses to induce and / or maintain immunity.

[0271] The vaccine should be administered in an "effective amount," meaning that the amount of the fusion protein and the STING agonist or a pharmaceutically acceptable salt thereof is sufficient, along the chosen route of administration, to elicit an immune response that effectively protects the host against the associated disease.

[0272] Representative diseases include (but are not limited to): influenza, viral pneumonia, viral pharyngitis, viral bronchitis, and bronchiolitis.

[0273] The amount of fusion protein selected in each vaccine dose is determined based on the amount that can elicit a protective immune response without significant side effects. Standards including antibody titers and other responses in the subjects can be used.

[0274] Research methods are used to determine the optimal dosage of a specific vaccine. Whether a booster dose is needed can be determined by monitoring the level of immunity provided by the vaccine. After assessing antibody titers in the serum, a booster dose may be required for immunization. Administration of adjuvants and / or immunostimulants can enhance the immune response to the proteins of this invention.

[0275] The preferred method is to administer the vaccine composition or immunogenic composition via a parenteral (intramuscular or intranasal) route.

[0276] Furthermore, the vaccine of the present invention can be administered in combination with other immunomodulators or with other therapeutic agents.

[0277] Preparation method

[0278] The fusion protein (or polypeptide) of the present invention can be recombinant or synthetic. The fusion protein of the present invention can be chemically synthesized or recombinant. Accordingly, the fusion protein of the present invention can be artificially synthesized using conventional methods or produced using recombinant methods.

[0279] A preferred method is to use liquid-phase synthesis or solid-phase synthesis techniques, such as the Boc solid-phase method, the Fmoc solid-phase method, or a combination of both. Solid-phase synthesis provides rapid sample preparation, and the appropriate resin support and synthesis system can be selected based on the sequence characteristics of the target peptide. For example, a preferred solid-phase support in the Fmoc system is Wang resin linked to the C-terminal amino acid of the peptide. The Wang resin structure is polystyrene, and the arm between the amino acid and the resin is 4-alkoxybenzyl alcohol. The peptide is treated with 25% hexahydropyridine / dimethylformamide at room temperature for 20 minutes to remove the Fmoc protecting group, and then extended sequentially from the C-terminus to the N-terminus according to the given amino acid sequence. After synthesis, the synthesized proinsulin-related peptide is cleaved from the resin with trifluoroacetic acid containing 4% p-methylphenol, and the protecting group is removed. The crude peptide can be obtained by filtration and ether precipitation after resin removal. The resulting product solution is lyophilized and then purified by gel filtration and reversed-phase high-performance liquid chromatography (HPLC). When using a Boc system for solid-phase synthesis, the preferred resin is a PAM resin linked to the C-terminal amino acid of the peptide. The PAM resin structure is polystyrene, and the arm between the amino acid and the PAM is 4-hydroxymethylphenylacetamide. In the Boc synthesis system, during the deprotection, neutralization, and coupling cycle, the protecting Boc group is removed with TFA / dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA / dichloromethane). After peptide chain condensation, the peptide chain is cleaved from the resin for 1 hour at 0°C with hydrogen fluoride (HF) containing p-cresol (5-10%), simultaneously removing the protecting group. The peptide is extracted with 50-80% acetic acid (containing a small amount of mercaptoethanol), and after lyophilization, further processed using Sephadex molecular sieves. The peptide is isolated and purified using G10 or Tsk-40f, followed by high-performance liquid chromatography (HPLC) purification to obtain the desired peptide. Various coupling agents and methods known in peptide chemistry can be used to couple the amino acid residues; for example, direct coupling can be performed using dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt), or 1,1,3,3-tetraureurylhexafluorophosphate (HBTU). The purity and structure of the synthesized short peptide can be confirmed by reversed-phase HPLC and mass spectrometry.

[0280] In one embodiment, the fusion protein of the present invention is prepared according to its sequence by solid-phase synthesis, purified by high-performance liquid chromatography to obtain a high-purity lyophilized peptide powder, which is then stored at -20°C.

[0281] Another method is to use recombinant technology to generate the fusion protein of this invention.

[0282] Since the fusion protein of this invention is relatively short, it is possible to consider tandemly linking multiple polypeptide elements together, recombinantly expressing them to obtain a multimeric expression product, and then forming the desired small peptide through methods such as enzyme digestion.

[0283] The main advantages of this invention include:

[0284] (1) This invention is the first to discover that activating the STING pathway can effectively upregulate the expression of OX40 on the surface of T cells. A novel combination application is proposed, in which STING agonists and OX40L fusion antigens can synergistically enhance the immune memory of induced vaccines.

[0285] (2) This invention provides a fusion protein of the receptor-binding domain (RBD) of the novel coronavirus, the IgG1 Fc fragment and the OX40 ligand (OX40L) fused together. The parts are connected in series by a linker arm so that they do not affect each other in structure. This ensures that the RBD is in the correct spatial structure and increases the immunogenicity.

[0286] (3) The vaccine composition of the present invention, which combines the fusion protein with the STING agonist, further significantly improves the levels of specific neutralizing antibodies and binding antibodies, and prolongs the duration of neutralizing antibodies and binding antibodies.

[0287] (4) The vaccine composition of the present invention promotes the proliferation of T cells (including Tfh) and maintains the survival of T cells, thereby enhancing the T cell immune response.

[0288] (5) The vaccine composition of the present invention effectively promotes the production of memory B cells and memory T cells, and significantly improves the long-term immune protection of the vaccine.

[0289] (6) The vaccine composition of the present invention can be applied to muscle immunity and can also promote immune response in synergistic with STING agonists through the mucosal immune pathway.

[0290] (7) The vaccine composition of the present invention also provides a new strategy for inducing broad-spectrum and long-lasting immune memory protection for other immunogens.

[0291] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0292] Materials and Methods

[0293] (1) Experimental Materials

[0294] Reagents: The high-purity RNA extraction kit (TransZol Up Plus RNA Kit) was purchased from TransGen Biotech Co., Ltd., Beijing. The 2-Step Real Time RT-PCR and Trizo were purchased from Takara Biotechnology (Beijing) Co., Ltd. The CD3+T cell magnetic bead sorting kit (MojoSort TM Mouse CD3 T Cell Isolation Kit) (Biolegend) was used. The immunoadjuvant cGAMP was purchased from Shanghai Aoyu Biotechnology Co., Ltd. The SARS-CoV-2 RBD-His (Delta) protein was purchased from Shanghai Kaika Biotech Co., Ltd. The Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP and other HRP secondary antibodies were purchased from Thermo Fisher Scientific (China) Co., Ltd. The RPMI 1640 medium was purchased from Shanghai Saiyi Biotechnology Co., Ltd. The fetal bovine serum (Gibico) was purchased from Thermo Fisher Scientific (China) Co., Ltd. The serum (Yeasen Biotech) was purchased from Yeasen Biotech Co., Ltd., Shanghai. The Luciferase kit and 5× cell lysis buffer were purchased from Promega Corporation.

[0295] Animals: 8-week-old female BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were housed in the SPF-class animal house of the School of Pharmacy, Fudan University. The experimental animal production license is SCXK (Hu) 2013-0016. The animal housing temperature was 20-24 °C and the humidity was 50 ± 5%. The feed and water were both sterilized and freely available to the animals. All animal experiments were carried out in accordance with the experimental animal management regulations of the School of Pharmacy, Fudan University.

[0296] Example 1: Effects of STING Agonist on Gene and Protein Expression of OX40 in T Cells

[0297] 1) Experimental Method

[0298] STING agonists CF501 (CAS No.:2408723-12-4) and cGAMP (CAS No.:849214-04-6) induce type I interferon-I (IFN-I) responses and the production of pro-inflammatory cytokines by activating STING (Cell research vol.32,3(2022):269-287.; The Journal of experimental medicine vol.215,5(2018):1287-1299.).

[0299] Therefore, the present invention uses a STING agonist (such as CF501 or cGAMP) as an immune adjuvant; in this embodiment, CF501 is used as an immune adjuvant.

[0300] The STING agonist CF501 (20 μg) was mixed with PBS to a final concentration of 244 μM. The mixture was injected intramuscularly into the lateral thigh of BALB / c mice in the CF501 group. The total volume of the mixture was 100 μL, with 50 μL injected into each lateral thigh. Mice in the PBS group were injected with only PBS.

[0301] Twenty-four hours after modeling, mice were sacrificed and inguinal lymph nodes were harvested. The inguinal lymph nodes were then ground through a 70 μm cell sieve, and single-cell suspensions were obtained using lymphocyte separation medium. The cells were incubated at 4°C for 30 minutes in the dark with PE-labeled anti-mouse CD134 (OX40) antibody and FITC-labeled anti-mouse CD3 antibody. The expression level of OX40 protein on the surface of T cells was detected by flow cytometry.

[0302] In addition, lymphocytes were isolated from healthy BALB / c mice. Mouse spleen tissue was ground through a 70μm cell sieve, and single-cell suspensions were obtained using lymphocyte separation medium. The single-cell suspensions were then adjusted to 2×10⁻⁶ cells / mL with RPMI 1640 medium containing 10% fetal bovine serum. 6 Cells were cultured at a concentration of [cells / mL] and seeded into 6-well plates, with 2 mL of cell suspension seeded into each well. STING agonist CF501 (final concentration 5 μM) and an equal volume of PBS were added to each well, mixed well, and incubated at 37°C in a 5% CO2 incubator for 6 hours. Subsequently, CD3 [cells / mL] were used for [cells / mL]. + T-cell magnetic bead sorting kit (catalog number: 480023) was used for positive sorting. Sorted CD3... +Total RNA was extracted from T cells using the TransZol Up Plus RNA kit. The extracted RNA was then reverse transcribed into cDNA using a Takara 2-Step Real Time RT-PCR kit. This cDNA was then used as a template for PCR experiments to detect the OX40 gene level.

[0303] 2) Experimental Results

[0304] The results showed that the STING agonist CF501 could increase CD3 levels in spleen cells in vitro. + OX40 gene expression level in T cells ( Figure 1 A) Following intramuscular injection of the STING agonist CF501, CD3+ was found in the inguinal lymph nodes of mice. + The expression level of OX40 protein on the surface of T cells increased synchronously. Figure 1 B) This study confirms that the STING agonist has a positive regulatory effect on OX40.

[0305] Example 2: Construction and expression of recombinant SARS-CoV-2-RBD-Fc-OX40L fusion protein

[0306] 1.1 Construction of SARS-CoV-2-RBD-Fc-OX40L

[0307] The spike protein (S) of SARS-CoV-2 is responsible for recognizing and invading cells. The receptor-binding domain (RBD) within the S protein is the most important, responsible for recognizing and binding to the angiotensin-converting enzyme 2 (ACE2) receptor to mediate viral entry into cells. Currently developed COVID-19 mRNA vaccines, protein subunit vaccines, and adenovirus vector vaccines are all developed targeting the SARS-CoV-2 S protein or RBD protein.

[0308] This invention uses the Delta variant of SARS-CoV-2, a respiratory virus, as an example. The receptor-binding domain (RBD), IgG1 Fc, and mouse-derived OX40L protein of SARS-CoV-2 are expressed in tandem via a linker arm. The structure of the fusion protein is as follows: Figure 2 As shown in the figure below.

[0309] In addition, in mammalian expression systems such as CHO cells and HEK293, the IL-2 signal peptide is often used to construct secretory expression vectors for therapeutic proteins such as antibody fragments and cytokines (under the guidance of the signal peptide, the newly synthesized protein enters the endoplasmic reticulum lumen. The signal peptide sequence is then cleaved by signal peptidase, so the fusion protein does not contain the IL-2 signal peptide after expression).

[0310] Therefore, after codon optimization of the constructed amino acid sequence, a gene fragment was synthesized, and ECORI and NheI restriction sites were added to both ends, respectively, and linked to the IL-2 signal peptide sequence at the front end, to construct the following... Figure 2 The pFUSE-hIgG1-Fc2 expression plasmid shown in the figure above.

[0311] SARS-CoV-2-RBD-Fc-OX40L (abbreviated as RBD-Fc-OX40L) includes the receptor-binding domain of SARS-CoV-2 (RBD of the Delta variant), IgG1 Fc, OX40L protein, and a linker arm located between IgG1 Fc and OX40L. The amino acid sequence of the linker arm is GGGGSGGGGSGGGGSGGGGS, as shown in SEQ ID NO.5.

[0312] 1.2 Expression of RBD-Fc-OX40L

[0313] 1) Experimental methods

[0314] (1) Passage the EXPi293T cells in good condition;

[0315] (2) After subculturing, wait until the cell density reaches 2.5 × 10⁻⁶. 6 The constructed fusion protein expression plasmid was transfected into EXPi293T cells using OPM-TR01 transfection reagent (OPM).

[0316] (3) After the cells were cultured for another 7 days, the supernatant of the suspended cells was collected, centrifuged at 3000 rpm and 4℃ for 30 minutes, and the supernatant was collected. Protein A Resin was used for protein purification, which included: adding 500 μL of Protein A Resin, equilibrating with PBS, adding protein supernatant and incubating for 2 hours, washing with 400 mL of PBS, eluting the target protein with 0.1 M glycine (pH=2.5) and equilibrating the pH to 7.8 with Tris-HCl solution;

[0317] (4) Divide the eluted protein into two tubes. Add 5× protein loading (containing β-mercaptoethanol) to one tube and boil at 100°C for 10 minutes. Add only 5× protein loading (without β-mercaptoethanol) to the other tube and do not boil. Then perform SDS-PAGE gel electrophoresis on the fusion protein.

[0318] 2) Experimental Results

[0319] like Figure 3As shown, the fusion protein RBD-Fc-OX40L was successfully expressed. The monomeric band of the boiled sample was about 80 kDa, while the band of the unboiled sample was about 160 kDa, indicating that the fusion protein RBD-Fc-OX40L stably formed a protein dimer under the action of Fc.

[0320] Example 3: Detection of the synergistic effect of STING agonist and fusion protein RBD-Fc-OX40L on promoting cytokine expression in mice after immunization

[0321] 1) Experimental methods

[0322] (1) Modeling: BALB / c mice were divided into three groups: PBS, RBD-Fc+CF501, and RBD-Fc-OX40L+CF501. In the RBD-Fc+CF501 and RBD-Fc-OX40L+CF501 treatment groups, the STING agonist CF501 (20 μg) and RBD-Fc or RBD-Fc-OX40L (5 μg) were mixed in PBS as solvent, with a total volume of 100 μL. BALB / c mice were injected intramuscularly into the lateral thigh of each thigh, with a volume of 100 μL, 50 μL injected into each thigh.

[0323] (2) Extraction of lymphocytes from mouse inguinal lymph nodes: Six hours after modeling, mice were sacrificed and inguinal lymph nodes were removed. The mouse inguinal lymph nodes were then ground through a 70μm cell sieve, and a single-cell suspension was obtained using lymphocyte separation fluid. RNA was extracted from the cells using the Trizol method, and cDNA was synthesized using the Takara two-step real-time reverse transcription PCR kit. The levels of genes such as IFN-γ were then detected.

[0324] (3) Extraction of T cells from mouse spleen cells in vitro: Lymphocytes were extracted from mouse spleens using the same method as in Example 1. Six hours after modeling, the cells were incubated with the STING agonist CF501 (final concentration 5 μM) and RBD-Fc or RBD-Fc-OX40L (5 μg) and CD3+. + Positive cells were selected using a T-cell magnetic bead sorting kit. Total RNA was extracted using the TransZol Up Plus RNA kit, and cDNA was synthesized using the Takara two-step real-time reverse transcription PCR kit for detection of genes such as BCL-XL.

[0325] 2) Experimental Results

[0326] like Figure 4As shown, fusion of OX40L protein with RBD-Fc followed by immunization with STING agonist further enhances the immunostimulatory effect of RBD-Fc in lymph nodes, and significantly increases the gene expression levels of inflammatory factors IFN-γ and IL-6.

[0327] Furthermore, the RBD-Fc-OX40L fusion protein significantly upregulated the anti-apoptotic protein BCL-XL in T cells from spleen cells in vitro and significantly downregulated the gene expression of the pro-apoptotic protein Caspase-3. This indicates that OX40L interacts with the OX40 protein in activated T cells, activating the OX40-OX40L pathway, thereby effectively inhibiting the T cell apoptosis pathway and promoting T cell survival. The results are shown in... Figure 5 .

[0328] The above results indicate that, compared to the combination of RBD-Fc protein and STING agonist, the combination of RBD-Fc-OX40L fusion protein and STING agonist significantly increased the expression of inflammatory factors and promoted T cell survival, exhibiting anti-apoptotic effects on T cells and thereby enhancing the level of immune response. Figure 4 and Figure 5 ).

[0329] Example 4: Detection of the synergistic effect of STING agonist and RBD-Fc-OX40L on promoting immune response in mice after muscle immunization (specifically binding antibody)

[0330] 1) Experimental methods

[0331] (1) Animal modeling

[0332] BALB / c mice were randomly divided into five groups: PBS, RBD-Fc treatment group, RBD-Fc-OX40L treatment group, RBD-Fc+CF501 treatment group, and RBD-Fc-OX40L+CF501 treatment group. In the RBD-Fc+CF501 and RBD-Fc-OX40L+CF501 treatment groups, the STING agonist CF501 (20 μg) and either RBD-Fc or RBD-Fc-OX40L (5 μg) were mixed in PBS to a total volume of 100 μL. In the RBD-Fc and RBD-Fc-OX40L groups, RBD-Fc or RBD-Fc-OX40L (5 μg) was mixed with PBS as a control. BALB / c mice were injected intramuscularly into the lateral thigh of each leg (50 μL in each leg). Immunization was performed in three doses, with the specific timing as follows: Figure 6 As shown.

[0333] Specific immunization procedures are as follows: Figure 6As shown, mice were immunized three times on days 0, 28, and 56, and blood was collected from the orbital venous plexus of mice on days 35 and 63. Mouse serum was separated and inactivated at 56°C for half an hour. The RBD-specific binding antibody of SARS-CoV-2 (Delta) strain in mouse serum was detected by ELISA.

[0334] (2) Enzyme-linked immunosorbent assay

[0335] (a) Dilute the SARS-CoV-2 RBD His protein to 1 μg / mL in carbonate buffer, then add 50 μL of the protein dilution to each well of an ELISA plate and coat the plate overnight at 4°C.

[0336] (b) On the second day, the ELISA plates were blocked with blocking buffer (PBST containing 5% BSA), 150 μL per well, and incubated at 37°C for 2 hours.

[0337] (c) Use PBST to perform three- or four-fold serial dilutions of mouse serum (100-fold dilution in the first well);

[0338] (d) Add 50 μL to each well of the ELISA plate and incubate at 37°C for 60 minutes.

[0339] (e) Wash four times with PBST and add enzyme-labeled secondary antibody. The enzyme-labeled secondary antibody is HRP-labeled goat anti-mouse IgG, IgG1, and IgG2a antibodies;

[0340] (f) After incubation at 37°C for 40 minutes, wash 4 times with PBST;

[0341] (g) After adding TMB substrate and developing color for 15 minutes, stop with dilute H2SO4, read the value at the detection wavelength of 450nm, and read the plate at the calibration wavelength of 630nm simultaneously.

[0342] 2) Experimental Results

[0343] The immune response of IgG antibodies in mouse serum 7 days after the second immunization (i.e., 35 days after immunization) was as follows: Figure 7 As shown in Table 1, the RBD-Fc-OX40L group and the RBD-Fc group produced comparable titers of RBD-specific IgG antibodies in mice; however, compared with the PBS group, the RBD-Fc-OX40L group and the RBD-Fc-OX40L+CF501 group produced significantly higher titers of RBD-specific IgG antibodies in mice.

[0344] Furthermore, unexpectedly, compared with the RBD-Fc+CF501 group and the RBD-Fc-OX40L group, the titer of RBD-specific IgG antibodies produced in mice in the RBD-Fc-OX40L+CF501 group was significantly increased 7 days after the second immunization (i.e., day 35 of immunization). In particular, compared with the RBD-Fc+CF501 group, the titer of specific IgG antibodies in the serum of mice in the RBD-Fc-OX40L+CF501 group was increased by about 3 times.

[0345] Table 1. Titer of SARS-CoV-2RBD (Delta variant) specific IgG antibody in the serum of mice 35 days after immunization

[0346]

[0347] *: Significantly different compared to RBD-Fc+CF501

[0348] In addition, the immune response of IgG, IgG1, and IgG2a antibodies in mouse serum 7 days after three immunizations was as follows: Figure 8 As shown in AC and Table 2-4, there was no significant difference in IgG antibody titers between the RBD-Fc-OX40L group and the RBD-Fc group.

[0349] However, unexpectedly, compared to the RBD-Fc+CF501 group and the RBD-Fc-OX40L group, the titer of RBD-specific IgG antibodies produced in the serum of mice 7 days after three immunizations with RBD-Fc-OX40L+CF501 (i.e., day 63 after immunization) was significantly increased (Table 2 and ). Figure 8 A), among which, compared with the RBD-Fc+CF501 group, the specific IgG antibody titer in the serum of mice in the RBD-Fc-OX40L+CF501 group increased by about 16 times on day 7 after three immunizations (i.e., day 63 of immunization).

[0350] In addition, IgG1 (Table 3 and ) was found in the serum of mice 7 days after three immunizations with RBD-Fc-OX40L. Figure 8 B) and IgG2a (Table 4 and Figure 8 The levels of C) were significantly increased, resulting in higher levels of binding antibodies; among them, compared with the RBD-Fc+CF501 group, the specific IgG1 antibody titer in the serum of mice in the RBD-Fc-OX40L+CF501 group increased by about 3.1 times and the IgG2a antibody titer increased by about 8.6 times after 7 days of three immunizations (i.e., day 63 of immunization).

[0351] The above results indicate that, when STING agonist was administered in combination with RBD-Fc-OX40L via intramuscular immunization, compared with RBD-Fc / CF501, the level of RBD-specific IgG antibodies in mice was significantly increased, and the class switching of IgG1 and IgG2a was promoted.

[0352] Table 2. SARS-CoV-2RBD (Delta variant) specific IgG antibody titers in the serum of mice 63 days after immunization

[0353]

[0354] *: Significantly different compared to RBD-Fc+CF501

[0355] Table 3. SARS-CoV-2RBD (Delta variant) specific IgG1 antibody titers in the serum of mice 63 days after immunization

[0356]

[0357] *: Significantly different compared to RBD-Fc+CF501

[0358] Table 4. SARS-CoV-2RBD (Delta variant) specific IgG2a antibody titers in the serum of mice 63 days after immunization

[0359]

[0360] *: Significantly different compared to RBD-Fc+CF501

[0361] Example 5: Detection of the synergistic effect of STING agonist and RBD-Fc-OX40L on promoting immune response in mice after muscle immunization (specific neutralizing antibody)

[0362] 1) Experimental methods

[0363] (1) Packaging of SARS-CoV-2 (Delta variants, etc.) and SARS-CoV pseudoviruses:

[0364] (a) Passage well-grown HEK-293T cells, and start transfection 24 hours after passage;

[0365] (b) HEK293T cells were co-transfected with plasmid pcDNA3.1-SARS-CoV-2-S (pcDNA3.1 plasmid containing Delta variant sequence) and pcDNA3.1-SARS-CoV-S with HIV backbone plasmid PNL-4-3-Luc using the transfection reagent Vigofect.

[0366] (c) Eight hours after transfection, the cells were replaced with fresh DMEM medium (containing 10% fetal bovine serum and 1× penicillin and streptomycin);

[0367] (d) After culturing for another 48 hours, the cell supernatant was collected, which contained SARS-CoV-2 (Delta variant, etc.) and SARS-CoV pseudovirus. After passing through a 0.45 μm filter, the cells were stored at -80 °C.

[0368] (2) Detection of neutralizing antibody titers against SARS-CoV-2 (Delta variant, etc.) and SARS-CoV pseudovirus in mouse serum:

[0369] (a) Using the same experimental animal modeling method as in Example 4, mouse serum was collected after treatment for subsequent testing;

[0370] (b) Huh-7 cells were seeded into 96-well plates, with 8000 cells per well;

[0371] (c) After 8 hours, begin diluting the serum by serially diluting it 3-4 times using DMEM (without 10% fetal bovine serum) (first well: 100-fold dilution). Include a virus control well, i.e., add only sham virus without mouse serum;

[0372] (d) Mix SARS-CoV-2 or SARS-CoV pseudovirus with diluted serum and incubate at 37°C for 30 minutes;

[0373] (e) Add to the prepared Huh-7 cells, and replace the cells with fresh DMEM medium containing 10% fetal bovine serum and 1× penicillin and streptomycin after 12 hours.

[0374] (f) After culturing for another 48 hours, the cells were lysed using the Promega Luciferase kit, and the activity of Luciferase was detected.

[0375] (g) Calculate the 50% neutralizing antibody titer in serum (NT). 50 value).

[0376] 2) Experimental Results

[0377] like Figure 9As shown in Table 5, compared with the RBD-Fc+CF501 group and the RBD-Fc-OX40L group, the neutralizing antibody titer against SARS-CoV-2 (Delta variant) pseudovirus in the serum of mice 7 days after the second intramuscular immunization with RBD-Fc-OX40L+CF501 (i.e., day 35 of immunization) was significantly increased; among them, compared with the RBD-Fc+CF501 group, the neutralizing antibody titer against SARS-CoV-2 (Delta variant) pseudovirus in the serum of mice in the RBD-Fc-OX40L+CF501 group 7 days after the second immunization (i.e., day 35 of immunization) was increased by about 1.6 times.

[0378] Table 5. Neutralizing antibodies against SARS-CoV-2 (Delta variant) pseudovirus in the serum of mice 35 days after immunization

[0379]

[0380] *: Significantly different compared to RBD-Fc+CF501

[0381] like Figure 10 As shown in A and Table 6, compared with the RBD-Fc+CF501 group and the RBD-Fc-OX40L group, the neutralizing antibody titer against SARS-CoV-2 (Delta variant) pseudovirus in the serum of mice 7 days after three intramuscular immunizations with RBD-Fc-OX40L+CF501 (i.e., day 63 of immunization) was significantly increased (to 60346.00±25205.40); among them, compared with the RBD-Fc+CF501 group, the neutralizing antibody titer against SARS-CoV-2 (Delta variant) pseudovirus in the serum of mice in the RBD-Fc-OX40L+CF501 group 7 days after three intramuscular immunizations (i.e., day 63 of immunization) was increased by about 2.5 times.

[0382] Table 6. Neutralizing antibodies against SARS-CoV-2 (Delta variant) pseudovirus in the serum of mice 63 days after immunization

[0383]

[0384] *: Significantly different compared to RBD-Fc+CF501

[0385] Furthermore, compared to mice in the RBD-Fc+CF501 group and the RBD-Fc-OX40L group, the serum of mice 7 days after three immunizations with RBD-Fc-OX40L+CF501 (i.e., day 63 of immunization) showed a higher seroprevalence against other SARS-CoV-2 variants, BA.2.2. Figure 10 B and Table 7) and BA.5 ( Figure 10C and Table 8), and the neutralizing antibody titer against SARS-CoV pseudovirus ( Figure 11 Both (as shown in Table 9) were significantly elevated; among them, compared with the RBD-Fc+CF501 group, the neutralizing antibody titer against SARS-CoV-2 (BA.2.2 variant) pseudovirus was increased by about 3.8 times, the neutralizing antibody titer against SARS-CoV-2 (BA.5 variant) pseudovirus was increased by about 2.9 times, and the neutralizing antibody titer against SARS-CoV pseudovirus was increased by 4.8 times in the serum of mice in the RBD-Fc-OX40L+CF501 group 7 days after three immunizations (i.e., day 63 of immunization).

[0386] This indicates that combining the RBD-Fc-OX40L fusion protein with the STING agonist for immunization, thereby further increasing the titer of neutralizing antibodies in mouse serum, is an effective immune-enhancing protective strategy.

[0387] Table 7. Neutralizing antibodies against SARS-CoV-2 (BA.2.2 variant) pseudovirus in the serum of mice 63 days after immunization

[0388]

[0389] *: Significantly different compared to RBD-Fc+CF501

[0390] Table 8. Neutralizing antibodies against SARS-CoV-2 (BA.5 variant) pseudovirus in the serum of mice 63 days after immunization

[0391]

[0392] *: Significantly different compared to RBD-Fc+CF501

[0393] Table 9. Neutralizing antibodies against SARS-CoV pseudovirus in the serum of mice 63 days after immunization

[0394]

[0395] *: Significantly different compared to RBD-Fc+CF501

[0396] Example 6: Flow cytometry detection of the synergistic promotion of follicular helper T cell (Tfh), GC B cell and memory B cell (MB) formation after immunization with combination of STING agonist and RBD-Fc-OX40L.

[0397] 1) Experimental methods

[0398] Using the same animal modeling method as in Example 4, on day 10 after three immunizations, inguinal lymph nodes and spleen tissues from different groups of mice were ground through a 70 μm cell sieve, and single-cell suspensions were obtained using lymphocyte separation fluid. The density of the spleen cell and inguinal lymph node cell suspensions was adjusted to 1 × 10⁻⁶. 8 cells / mL. Cells were stained according to different staining protocols for Tfh, GC B, and MB.

[0399] Flow cytometry sample tubes were then prepared. Flow cytometry antibodies targeting Tfh in inguinal lymph nodes included anti-mouse CD3, CD4, PD-1, and CXCR5 antibodies. Flow cytometry antibodies targeting GC B cells in inguinal lymph nodes included anti-mouse B220 and GL-7 antibodies. Flow cytometry antibodies targeting memory B cells in the spleen included anti-mouse CD3, CD38, IgD, SARS-CoV-2-RBD (Delta variant)-PE, and SARS-CoV-2-RBD (Delta variant)-APC antibodies (where RBD-PE and RBD-APC were obtained by incubating the biotinylated SARS-CoV-2-RBD-His protein with PE or APC flow cytometry fluorescent antibodies).

[0400] Mouse flow cytometry samples were stained using the different types of antibodies described above, and then cell phenotypes were identified using Beckman flow cytometry to detect Tfh globular clusters (CD3+). + CD4 + PD-1 + CXCR5 + ), detection of GC B cell spheroids (B220) + GL-7 + ), detection of MB cells (i.e., memory B cells) gate (CD3) + CD38 + IgD - RB D-PE + RBD-APC + ).

[0401] 2) Experimental Results

[0402] The results are as follows Figure 12 As shown, compared to the PBS group, mice immunized three times with RBD-Fc+CF501 and RBD-Fc-OX40L+CF501, respectively, showed elevated levels of Tfh ( ) in the lymph nodes of mice in both groups on day 10. Figure 12 A) and GC B( Figure 12 B), and specific memory B cells in spleen tissue (B), Figure 12 C) all increased significantly.

[0403] However, unexpectedly, the proportions of Tfh cells, GC B cells, and specific memory B cells in the lymph nodes and spleen tissue of mice treated with RBD-RBD-Fc-OX40L+CF501 were significantly higher than those in the RBD-RBD-Fc+CF501 treatment group. This indicates that after the STING agonist activates the expression of OX40 on the surface of T cells, OX40L in the fusion protein RBD-Fc-OX40L may bind to the activated OX40 on the surface of T cells, activate the OX40-OX40L pathway, promote the proliferation of T cells (such as Tfh cells), and further promote the formation of GC B cells and the differentiation of memory B cells. The above results are consistent with the antibody level detection results of Examples 4 and 5.

[0404] Example 7: Long-term assay of the synergistic effect of STING agonist combined with RBD-Fc-OX40L on promoting immune response after muscle immunization (binding antibody)

[0405] 1) Experimental methods

[0406] (1) Animal modeling

[0407] BALB / c mice were divided into two groups: the RBD-Fc+CF501 treatment group and the RBD-Fc-OX40L+CF501 treatment group. Animal modeling was performed using the same method as in Example 4.

[0408] Specific immunization procedures are as follows: Figure 6 As shown, mice were immunized three times on days 0, 28, and 56. Blood from the orbital venous plexus of mice was collected on day 30 (one month) and day 180 (six months) after the third immunization. Mouse serum was separated and inactivated at 56°C for half an hour. The SARS-CoV-2 RBD-specific binding antibody in the mouse serum was detected by ELISA.

[0409] (2) Enzyme-linked immunosorbent assay: The enzyme-linked immunosorbent assay was performed using the same method as in Example 4.

[0410] 2) Experimental Results

[0411] The results showed that, compared with the positive control group RBD-Fc+CF501 treated mice, on day 30 after three immunizations with RBD-Fc-OX40L+CF501 (i.e., one month after immunization)... Figure 13 (A and Table 10) and day 180 (i.e., six months after immunization) Figure 13The serum of mice in groups B and (Table 11) still maintained a high level of RBD-specific IgG antibodies, which were significantly higher than those in the RBD-Fc+CF501 treatment group. This indicates that the combination of STING agonist and RBD-Fc-OX40L can effectively prolong the duration of the immune response.

[0412] Table 10. Serum titers of SARS-CoV-2RBD (Delta variant) specific IgG antibodies in mice on day 30 (one month) after three immunizations (mean ± standard deviation)

[0413]

[0414] Table 11. Serum titers of SARS-CoV-2RBD (Delta variant) specific IgG antibodies in mice on day 180 (six months) after three immunizations (mean ± standard deviation)

[0415]

[0416] Example 8: Long-term assay of the synergistic effect of STING agonist combined with RBD-Fc-OX40L on promoting immune response after muscle immunization (specific neutralizing antibody)

[0417] 1) Experimental methods

[0418] (1) Packaging of SARS-CoV-2 (Delta variant) pseudovirus:

[0419] In this embodiment, the same pseudovirus packaging experiment method as in Example 5 was used to co-transfect HEK293T cells with plasmid pcDNA3.1-SARS-CoV-2-S (Delta variant) and HIV backbone plasmid PNL-4-3-Luc, followed by subsequent culture and collection of SARS-CoV-2 pseudoviruses.

[0420] (2) The same method as experimental method (2) in Example 5 was used to detect SARS-CoV-2 (Delta variant) pseudovirus in mouse serum.

[0421] 2) Experimental Results

[0422] The results showed that, compared with the RBD-Fc+CF501 treatment group, mice treated with RBD-Fc-OX40L+CF501 showed improved remission rates on day 30 (one month) after three immunizations. Figure 14 A and Table 12) and day 180 (six months) Figure 14The neutralizing antibody titers against SARS-CoV-2 (Delta variant) pseudovirus in the serum of mice in B and Table 13) were significantly increased. Table 12. Neutralizing antibody titers against SARS-CoV-2 (Delta) pseudovirus in mouse serum on day 30 (one month) after three immunizations.

[0423]

[0424] Table 13. Neutralizing antibody titers against SARS-CoV-2 (Delta) pseudovirus in mouse serum on day 180 (six months) after three immunizations.

[0425]

[0426] Example 9: Detection of the synergistic effect of STING agonist and RBD-Fc-OX40L on promoting immune response in mice after mucosal immunization (specifically binding antibody)

[0427] 1) Experimental methods

[0428] In this embodiment, cGAMP is used as a mucosal immune adjuvant.

[0429] BALB / c mice were divided into two groups: RBD-Fc+cGAMP and RBD-Fc-OX40L+cGAMP. The STING agonist cGAMP (20 μg) was mixed with RBD-Fc or RBD-Fc-OX40L (5 μg) using PBS as a solvent. The total volume of the mixture was 20 μL.

[0430] After anesthetizing BALB / c mice, the mixed solution was instilled into the nasal cavity of the mice, with each administration volume being 20 μL, divided into three immunizations. The specific immunization time is as follows. Figure 6 As shown.

[0431] Specific immunization procedures are as follows: Figure 6 As shown, mice were immunized three times on days 0, 28, and 56, and blood was collected from the orbital venous plexus of mice on days 35 and 63. Mouse serum was separated and inactivated at 56°C for half an hour. The SARS-CoV-2 RBD-specific binding antibody in the mouse serum was detected by ELISA.

[0432] (2) Enzyme-linked immunosorbent assay: The enzyme-linked immunosorbent assay was performed using the same method as in Example 4, wherein the enzyme-labeled secondary antibodies were HRP-labeled goat anti-mouse IgG and IgA.

[0433] 2) Experimental Results

[0434] The results showed that, compared with the RBD-Fc+cGAMP treatment group, the serum IgG antibody levels in mice 7 days after the second immunization (i.e., day 35 after immunization) were significantly higher. Figure 15 A and Table 14) and IgA antibody ( Figure 15 (B and Table 15) Immune response: After two immunizations with RBD-Fc-OX40L+cGAMP, the levels of RBD-specific IgG and IgA antibodies against SARS-CoV-2 in mouse serum were significantly increased.

[0435] Table 14. SARS-CoV-2RBD (Delta variant) specific IgG antibody titers in the serum of mice 35 days after mucosal immunization

[0436]

[0437] Table 15. SARS-CoV-2RBD (Delta variant) specific IgA antibody titers in mouse serum 35 days after mucosal immunization

[0438]

[0439] Furthermore, compared to mice in the positive control group treated with RBD-Fc+cGAMP, mice 7 days after being immunized three times with RBD-Fc-OX40L+cGAMP showed higher levels of RBD-specific IgG against SARS-CoV-2 in their serum. Figure 16 A and Table 16) and IgA ( Figure 16 B and Table 17) showed a significant increase in antibody titers.

[0440] Table 16. SARS-CoV-2RBD (Delta variant) specific IgG antibody titers in the serum of mice 63 days after mucosal immunization

[0441]

[0442] Table 17. SARS-CoV-2RBD (Delta variant) specific IgA antibody titers in the serum of mice 63 days after mucosal immunization

[0443]

[0444] Furthermore, surprisingly, compared to the RBD-Fc+CF501 group, mice that underwent a second immunization via intramuscular injection of a mixture of RBD-Fc-OX40L and CF501 seven days later (i.e., day 35 of immunization) showed an approximately 3-fold increase in serum IgG antibodies; while compared to the RBD-Fc+cGAMP group, mice that underwent mucosal immunization with a mixture of RBD-Fc-OX40L and cGAMP seven days later (i.e., day 35 of immunization) showed an approximately 22.9-fold increase in serum IgG antibodies.

[0445] For mice immunized for 63 days, compared with the RBD-Fc+CF501 group, the serum specific IgG antibody titer of mice immunized by intramuscular injection after mixing RBD-Fc-OX40L and CF501 increased by about 16 times; and compared with the RBD-Fc+cGAMP group, the serum specific IgG antibody titer of mice immunized by mucosal immunization after mixing RBD-Fc-OX40L and cGAMP increased by about 12.3 times.

[0446] Example 10: Detection of the synergistic effect of STING agonist and RBD-Fc-OX40L on promoting immune response in mice after mucosal immunization (specific neutralizing antibody)

[0447] 1) Experimental methods

[0448] (1) Packaging of SARS-CoV-2 (Delta variant) pseudovirus:

[0449] In this embodiment, the same pseudovirus packaging experiment method as in Example 5 was used to co-transfect HEK293T cells with plasmid pcDNA3.1-SARS-CoV-2-S (Delta variant) and HIV backbone plasmid PNL-4-3-Luc, followed by subsequent culture and collection of SARS-CoV-2 pseudoviruses.

[0450] (2) Detection of SARS-CoV-2 (Delta variant) pseudoviruses in mouse serum:

[0451] (a) Using the same experimental animal modeling method as in Example 9, mouse serum was collected after treatment for subsequent testing;

[0452] (b) The same method as experimental method (2) in Example 5 was used to detect SARS-CoV-2 (Delta variant) pseudoviruses in mouse serum.

[0453] 2) Experimental Results

[0454] like Figure 17 As shown in A and Table 18, compared with the control group of RBD-Fc+cGAMP mice, the neutralizing antibody titer against SARS-CoV-2 (Delta variant) pseudovirus was significantly increased in the serum of mice 7 days after two mucosal immunizations with RBD-Fc-OX40L+cGAMP (i.e., day 35 of immunization). This indicates that RBD-Fc-OX40L+cGAMP produces higher levels of neutralizing antibodies in mice after two mucosal immunizations.

[0455] Table 18. Neutralizing antibodies against SARS-CoV-2 (Delta variant) pseudovirus in the serum of mice immunized with mucosa for 35 days.

[0456]

[0457] like Figure 17 As shown in B and Table 19, compared with the control group of RBD-Fc+cGAMP mice, the neutralizing antibody titer against SARS-CoV-2 (Delta variant) pseudovirus was significantly increased in the serum of mice 7 days after three mucosal immunizations with RBD-Fc-OX40L+cGAMP (i.e., day 63 of immunization).

[0458] This indicates that combining the RBD-Fc-OX40L fusion protein with the STING agonist for mucosal immunization can further increase the neutralizing antibody titer in mouse serum, which is an effective mucosal immune enhancement and protection strategy.

[0459] Table 19. Neutralizing antibodies against SARS-CoV-2 (Delta variant) pseudovirus in the serum of mice immunized with mucosa for 63 days.

[0460]

[0461] discuss

[0462] The STING pathway, as an important signal transduction hub in the innate immune system, can regulate vaccine-induced immune persistence through multidimensional mechanisms. Currently, several novel STING agonist adjuvants, such as cGAMP and CF501, have been reported, aiming to prolong and enhance the production time, intensity, and duration of IFN-I, achieving long-term activation of STING and thus promoting the generation of immune memory (Cell research vol.32,3(2022):269-287.; Science (New York, NY) vol.369,6506(2020):eaba6098.). STING agonists, such as cGAMP, specifically bind to the STING protein on the endoplasmic reticulum, triggering a TBK1 kinase phosphorylation cascade, promoting the nuclear translocation of interferon regulatory factor 3 (IRF3), and initiating the sustained secretion of type I interferon (IFN-I), providing a key initiation signal for the immune response (Medicinal research reviews vol.44,4(2024):1768-1799.). Furthermore, the STING pathway can enhance adaptive immune responses through various mechanisms. For example, secreted type I interferon can directly enhance CD8... + The cytotoxic effect of T cells promotes the differentiation of memory T cells. Moreover, by secreting chemokines such as CXCL10, T cells are encouraged to hom to secondary lymphoid organs, which helps to form stable immune synapses and prolong the duration of the immune response (Immunity vol.55,2(2022):308-323.e9.).

[0463] OX40 is a member of the tumor necrosis factor receptor (TNFR) family, also known as CD134 or TNFRSF4, and is primarily expressed on activated CD4+. + and CD8 + On the surface of T cells, their ligand OX40L (CD252) is enriched in antigen-presenting cells (such as dendritic cells). The OX40-OX40L pathway plays a crucial role in inducing immune memory, primarily by promoting T cell proliferation, differentiation, and maintaining T cell survival to enhance the production of memory T cells (The Journal of Experimental Medicine vol. 191, 2(2000): 365-74.). First, OX40 significantly promotes T cell clonal expansion by activating the PI3K-Akt pathway and inducing the release of cytokine cascades such as IL-2 and IFN-γ. Then, the OX40 signaling pathway activates the NF-κB and PI3K / AKT pathways, upregulating the expression of anti-apoptotic proteins and cell cycle progression proteins, thereby effectively inhibiting apoptosis and promoting T cell survival. Finally, OX40, through the secretion of cytokines such as IFN-γ, promotes the differentiation of effector T cells into memory T cells while maintaining their long-term survival. Furthermore, activation of the OX40 pathway contributes to the formation of memory B cells. Follicular helper T cells (Tfh) play a crucial role in the formation of memory B cells. Tfh can promote the formation of GC B cells. The OX40 pathway plays an important role in the differentiation of Tfh cells by upregulating CXCR-5 and BCL-6 and downregulating PRDM1 transcription factor.

[0464] The vaccine compositions of the present invention (such as the combination of the fusion protein and the STING agonist of the present invention) can achieve enhanced and long-lasting immune effects.

[0465] In summary, the vaccine composition of the present invention comprises the OX40L fusion protein of the present invention and the STING agonist. The STING agonist activates the STING signaling pathway, effectively upregulating the OX40 protein on the surface of T cells. The STING agonist interacts with the OX40L on the fusion protein, activating the OX40 signaling pathway, significantly increasing the levels of specific neutralizing antibodies and binding antibodies, and prolonging the duration of neutralizing and binding antibodies. This promotes the differentiation of memory B cells and enhances the long-term protective effect of humoral immunity. Furthermore, the vaccine composition of the present invention can maintain the survival of T cells and promote the differentiation of T cells into immune memory cells, significantly enhancing the long-term immune response of T cells, thereby achieving preventive and / or therapeutic effects.

[0466] Finally, the vaccine composition of the present invention can also increase the levels of heterologous neutralizing antibodies and binding antibodies in serum through the mucosal immune pathway, and is also an effective long-term protective strategy for enhancing mucosal immunity, with broad application prospects.

[0467] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0468] The sequence information of this invention is shown in Table A.

[0469] Table A

[0470]

[0471]

Claims

1. A fusion protein, characterized in that, The fusion protein includes the following elements fused together: (a) Respiratory pathogen antigenic elements; (b) Fc fragment; and (c) OX40L or its active fragment.

2. The fusion protein as described in claim 1, characterized in that, The fusion protein has the structure shown in Formula I: Z1-Z2-L-Z3(I) In the formula, Z1 is a respiratory pathogen antigen element (preferably, a receptor domain RBD fragment element of the respiratory virus SARS-CoV-2). Z2 is the Fc fragment of IgG. L represents a linker peptide element or is absent; and Z3 is an OX40L component. "-" represents a peptide bond or a linking peptide independently.

3. A combination of active ingredients, characterized in that, The components of the combination include: (a) the fusion protein of claim 1 or 2 or its coding sequence or its expression vector and (b) an adjuvant, wherein the adjuvant is a STING agonist.

4. A pharmaceutical composition, characterized in that, include: (a) The fusion protein as described in claim 1 or 2, or its coding sequence or its expression vector, as an active ingredient; (b) Pharmaceutically acceptable carriers.

5. A polynucleotide, characterized in that, The polynucleotide encodes the fusion protein as described in claim 1 or 2.

6. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 5.

7. A host cell, characterized in that, The host cell contains the vector of claim 6 or the genome is integrated with the polynucleotide of claim 5.

8. Use of a combination of active ingredients as described in claim 3, characterized in that, Used to prepare vaccine compositions or vaccines for the prevention and / or treatment of diseases induced by respiratory tract infections, wherein, The vaccine composition or vaccine comprises: (i) the fusion protein as described in claim 1 or 2, or its coding sequence or expression vector, as an active ingredient, and (ii) the STING agonist or a pharmaceutically acceptable salt thereof.

9. A method for preparing the fusion protein, characterized in that, Includes the following steps: (i) Under suitable expression conditions, the host cells as described in claim 7 are cultured to obtain a culture medium containing the expressed protein; (ii) The fusion protein is isolated from the culture medium to obtain the isolated fusion protein.

10. A medicine box, characterized in that, The kit contains: (A) a first container, and a fusion protein as described in claim 1 or 2, a polynucleotide as described in claim 5, or a vector as described in claim 6, located within the first container; and (B) A second container, and a STING agonist or a pharmaceutically acceptable salt thereof located in the second container.

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