A fusion protein capable of promoting secretion of cell membrane fixed protein, a recombinant vaccine and its application

Abstract

The present invention relates to a vaccine framework fusion protein, a recombinant vaccine and its application. The fusion protein of the present invention comprises the intracellular segment of the low-affinity immunoglobulin gamma Fc region receptor II (FcγRII) and the WW domain (WW domain) of the E3 ubiquitin protein ligase Nedd4 family member protein, and / or one or more antigens, and / or one or more transmembrane regions. The present invention provides a new recombinant vaccine molecular architecture, which has a better ability to promote the expression and secretion of immune antigens and improve the effect of immune level, provides new ideas and methods for the design of recombinant vaccines, and can be widely used in the field of vaccines.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, in particular to the field of humoral immunity, and specifically to a humoral immunity vaccine molecular architecture comprising an FcγRII intracellular segment and a Nedd4 WW domain and an application thereof. Background Art

[0002] Humoral immunity is an important part of the immune system and is mainly mediated and regulated by B cells. When the body is invaded by pathogens (such as bacteria and viruses), the antigen is recognized and internalized by the receptor (BCR) on the surface of B cells, degraded into peptides in the cell and presented to helper T cells. Activated helper T cells provide co-stimulatory signals to B cells, promoting B cell proliferation and differentiation to produce a large number of antibodies against the antigen. These antibodies can eliminate pathogens by neutralization, precipitation, complement activation, and manipulation of killer cells. At the same time, a small number of B cells differentiate into memory B cells to provide long-term immune protection for the body.

[0003] Humoral immunity vaccines refer to vaccines that can induce the body to produce humoral immune responses and are the main type of preventive vaccines. Traditional humoral immunity vaccines include inactivated vaccines, live attenuated vaccines and recombinant protein vaccines, which stimulate the body to produce targeted antibodies through the antigen molecules in the vaccine. In recent years, nucleic acid vaccines, as a new type of humoral immunity vaccine, have shown great development potential.

[0004] Nucleic acid vaccines refer to DNA or mRNA molecular vaccines that carry viral antigen genes. After these nucleic acid molecules are expressed in the body, the antigenic proteins produced can induce humoral and cellular immune responses. Compared with traditional vaccines, nucleic acid vaccines have the advantages of good safety, easy large-scale preparation, and rapid design for new viruses. They have broad application prospects in the prevention and treatment of pathogen-infected diseases.

[0005] Exogenous membrane proteins and secretory proteins are important types of humoral immune antigens. However, conventional humoral immune nucleic acid vaccines often have difficulty expressing these target proteins efficiently, and the target proteins cannot be fully presented outside the cells, and the probability of being effectively recognized by B cell receptors is low. Therefore, they can only induce the body to produce low levels of antibodies, and the immune protection effect is limited. Therefore, increasing the expression level of humoral immune antigens and effectively presenting them to immune organs or germinal centers enriched with B cells is a key step in improving the immunogenicity of such vaccines.

[0006] Exosomes are vesicles (EVs) produced and secreted by living cells. They are 30-160nm in size and have a double-layer membrane structure like the cell membrane, which participates in the transfer of molecules between cells. Almost all cells can produce exosomes. At present, there are studies that use the characteristics of exosomes for artificial modification so that exosomes can be used in the field of drug delivery. For example, the prior art Chinese patents CN115708871A (publication date 2023-02-24) and CN115737796A (2023-03-07) disclose a method for presenting antigens on the surface of EVs. This method fuses the antigen to the extracellular domain of the CD63 protein, so that the EVs containing CD63 carry the antigen when budding, inducing the body to produce an immune response. However, since most of the EVs carrying CD63 are located inside the cell and the number that can be secreted outside the cell is limited, the induced humoral immunity is not at the optimal level. In addition, this method fuses the antigen between the two transmembrane regions of CD63, which may affect the conformation of the antigen protein and hinder the function of the conformational epitope.

[0007] Arrestin Domain Containing 1 (ARRDC1)-mediated protein endocytosis can form microvesicles, which are usually associated with G protein-coupled receptor internalization and play a role in regulating the internalization cycle of various membrane proteins such as receptors, transporters and ion channels. At present, studies have used this protein to produce recombinant exosomes. For example, US Patent US11730823B2 discloses a method that can load RNA into microvesicles (ARMM) mediated by ARRDC1. This method fuses RNA binding protein to the C-terminus of ARRDC1 protein, thereby loading specific RNA into ARMM to become an RNA drug carrier. Because ARRDC1 has a large molecular weight and is mainly related to cell endocytosis and internalization pathways, it has been proven by many studies that it is not conducive to the presentation of antigens on the membrane surface, and therefore cannot be used in the development of humoral immune vaccines.

[0008] The C-terminus of some E3 ubiquitin protein ligase Nedd4 family members contains a WW domain, which is composed of four elements: WW1, WW2, WW3, and WW4. Nedd4 is a gene that was originally found to be highly expressed in the central nervous system of early mouse embryos. Subsequently, the NEDD4-like protein family was identified in humans to include 7 members. NEDD4 and NEDD4L proteins include multiple functional domains, including calcium-dependent phospholipid and membrane binding domains (C2 domains), two to four protein binding domains (WW domains), and an E3 ubiquitin-protein ligase domain (HECT domain). Patent US20190374506A1 (published on 2019-12-12) discloses a method for treating cancer, which includes administering to a subject suffering from cancer an effective amount of an agent that inhibits the expression or activity of developmentally down-regulated protein 4 (NEDD4) expressed in neural precursor cells, and an agent that inhibits the expression or activity of WW domain protein 1 (WWP1). Low affinity immunoglobulin gamma Fc region receptor II (FcγRII) is a receptor widely expressed on the surface of human cells. It belongs to the immunoglobulin G (IgG) Fc region receptor family and is mainly responsible for regulating the activation and inhibition of immune cells. FcγRII is a transmembrane protein consisting of an extracellular region, a transmembrane region, and an intracellular region. Studies have been conducted to fuse its extracellular region with a target protein. For example, patent WO2022158836A1 (published on 2022-07-28) discloses a recombinant exosome comprising an extracellular segment or a portion thereof of an Fc receptor that can be fused with a target protein or peptide, wherein the Fc receptor includes FcγRII. Patent CN116782932A (published on 2023-09-19) discloses a composition of extracellular vesicles comprising a target protein fused with an extracellular segment of an Fc receptor, wherein the Fc receptor includes FcγRII. However, no prior art has yet disclosed the content related to the intracellular segment of FcγRII and exosomes.

[0009] Therefore, the field still urgently needs a new fusion antigen architecture that is more conducive to the secretion of cell membrane-fixed proteins, as well as a correspondingly constructed recombinant vaccine, which can achieve better ability to promote the expression and secretion of humoral immune antigens and improve the level of humoral immunity without destroying the conformation of humoral immune antigens. Summary of the invention

[0010] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a humoral immune vaccine framework fusion protein, recombinant vaccine, etc. The fusion protein of the present invention comprises the intracellular segment of the low-affinity immunoglobulin γ Fc region receptor II (FcγRII), the WW domain (WW domain) of the E3 ubiquitin protein ligase Nedd4 family member protein, and / or one or more antigens, and / or one or more transmembrane regions. The present invention provides a new recombinant vaccine molecular architecture, which has a better ability to promote the expression and secretion of humoral immune antigens and improve the effect of humoral immunity level, and provides new ideas and methods for the design of recombinant vaccines, which can be widely used in the field of humoral immune vaccines. The present invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, pharmaceutical uses, application methods, etc.

[0011] One aspect of the present invention provides a fusion protein, characterized in that it comprises the following elements:

[0012] (a) the intracellular region of the low affinity immunoglobulin gamma Fc region receptor II (FcγRII); and

[0013] (b) WW domain of E3 ubiquitin protein ligase Nedd4 family member proteins.

[0014] Furthermore, it is characterized in that the fusion protein also contains (c) one or more antigens; preferably, the antigen is a complete sequence of a humoral immune protein or a sequence that partially contains a humoral immune epitope; more preferably, the humoral immune protein is a membrane protein or a secretory protein of a pathogen; most preferably, the membrane protein or secretory protein of the pathogen is a partial or full-length sequence of the HA protein of avian influenza or the E2 protein of BVDV (bovine viral diarrhea virus).

[0015] Furthermore, it is characterized in that the antigen is the HA protein of avian influenza H9N2, the E2 protein of BVDV (bovine viral diarrhea virus), or the HA protein of avian influenza H1N1; preferably, the amino acid sequence of the HA protein of avian influenza H9N2 is shown in SEQ ID NO:5, the amino acid sequence of the E2 protein of BVDV is shown in SEQ ID NO:6, and the amino acid sequence of the HA protein of avian influenza H1N1 is shown in SEQ ID NO:7.

[0016] Furthermore, it is characterized in that the fusion protein also comprises (d) one or more transmembrane regions; preferably, the transmembrane region can be the antigen sequence's own transmembrane motif, or a transmembrane motif truncated from other membrane proteins; more preferably, the transmembrane motif truncated from other membrane proteins comes from the CD80 protein of any species; most preferably, the amino acid sequence of the transmembrane motif of the CD80 protein is as shown in SEQ ID NO:15, and the amino acid sequence of the self-existing transmembrane motif is as shown in SEQ ID NO:13.

[0017] Furthermore, it is characterized in that the intracellular segment of FcγRII can be the full-length or partial sequence of the cytoplasmic region; preferably, the intracellular segment of FcγRII is derived from mouse, guinea pig or human; more preferably, the amino acid sequence of the intracellular segment of FcγRII derived from mouse is as shown in SEQ ID NO: 3, or the amino acid sequence of the intracellular segment of FcγRII derived from guinea pig is as shown in SEQ ID NO: 4, or the intracellular segment of FcγRII is derived from human FcγRIIa, and the amino acid sequence of the intracellular segment of human FcγRIIa is as shown in SEQ ID NO: 11.

[0018] Furthermore, it is characterized in that the WW domain is located on the cytoplasmic side, and its amino acid sequence is shown in SEQ ID NO: 2.

[0019] Furthermore, it is characterized in that the various parts of the constituent elements can be optionally separated by a linker sequence; preferably, the linker sequence is shown in SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10.

[0020] Furthermore, it is characterized in that the fusion protein comprises the following elements in sequence from the N-terminus to the C-terminus:

[0021] (1) The antigen; preferably, the antigen is the HA protein of avian influenza H9N2 with an amino acid sequence as shown in SEQ ID NO:5, the E2 protein of BVDV with an amino acid sequence as shown in SEQ ID NO:6, or the HA protein of avian influenza H1N1 with an amino acid sequence as shown in SEQ ID NO:7;

[0022] (2) The transmembrane region; preferably, the transmembrane region comprises a transmembrane motif, and the transmembrane motif is a transmembrane motif of the antigen sequence itself, or a CD80 protein from any species; more preferably, the amino acid sequence of the transmembrane motif of the CD80 protein is as shown in SEQ ID NO: 15;

[0023] (3) The intracellular segment of FcγRII; preferably, the intracellular segment of FcγRII is the intracellular segment of FcγRII derived from mouse, whose amino acid sequence is shown in SEQ ID NO: 3, or the intracellular segment of FcγRII derived from guinea pig, whose amino acid sequence is shown in SEQ ID NO: 4.

[0024] (4) The WW domain; preferably, the amino acid sequence of the WW domain is as shown in SEQ ID NO: 2.

[0025] The various parts constituting the elements (1-(4)) may be optionally separated by a linker sequence. Preferably, the linker sequence is as shown in SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.

[0026] Furthermore, it is characterized in that the fusion protein also contains a signal peptide sequence; preferably, the amino acid sequence of the signal peptide sequence is as shown in SEQ ID NO:12 or SEQ ID NO:14.

[0027] Furthermore, it is characterized in that the fusion protein is any one of the following:

[0028] (A) From N-terminus to C-terminus, it contains the following elements: target antigen 1 with an amino acid sequence as shown in SEQ ID NO: 5, mouse FcγRII intracellular segment with an amino acid sequence as shown in SEQ ID NO: 3, and WW domain with an amino acid sequence as shown in SEQ ID NO: 2;

[0029] (B) from N-terminus to C-terminus, it comprises the following elements: a target antigen 1 having an amino acid sequence as shown in SEQ ID NO: 5, an intracellular segment of guinea pig FcγRII having an amino acid sequence as shown in SEQ ID NO: 4, and a WWdomain having an amino acid sequence as shown in SEQ ID NO: 2;

[0030] (C) a signal peptide having an amino acid sequence as shown in SEQ ID NO: 14, a target antigen 2 having an amino acid sequence as shown in SEQ ID NO: 6, a transmembrane region having an amino acid sequence as shown in SEQ ID NO: 15, a guinea pig FcγRII intracellular segment having an amino acid sequence as shown in SEQ ID NO: 4, and a WW domain having an amino acid sequence as shown in SEQ ID NO: 2;

[0031] The various parts of the elements may optionally be separated by linker sequences.

[0032] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it encodes the fusion protein of the present invention.

[0033] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule of the present invention.

[0034] Furthermore, it is characterized in that the recombinant gene expression cassette comprises the following elements in sequence from the 5' end to the 3' end: a promoter, a 5' UTR, a nucleic acid encoding a signal peptide, an antigen nucleic acid sequence, the transmembrane region, the intracellular segment of the FcγRII, the WWdomain, a 3' UTR, and a PolyA tail sequence; wherein the various parts of the elements can be optionally separated by a linker sequence.

[0035] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule or the recombinant gene expression cassette of the present invention.

[0036] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule, the recombinant gene expression cassette, or the recombinant vector of the present invention.

[0037] Another aspect of the present invention provides a pharmaceutical composition, characterized in that it contains one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention.

[0038] Furthermore, it is characterized in that the pharmaceutical composition also contains a pharmaceutically acceptable carrier.

[0039] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more fusion proteins described in the present invention, and / or one or more recombinant nucleic acid molecules described in the present invention, and / or one or more recombinant gene expression cassettes described in the present invention, and / or one or more recombinant vectors described in the present invention, and / or one or more recombinant host cells described in the present invention.

[0040] Another aspect of the present invention provides a method for promoting the secretion of fusion proteins outside cells in the form of exosomes, characterized in that the method comprises administering one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more recombinant vaccines of the present invention to cells and / or other organisms; preferably, the method is for non-therapeutic and diagnostic purposes.

[0041] Another aspect of the present invention provides a method for improving the immunogenicity of humoral immune antigens, characterized in that the method comprises administering one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more recombinant vaccines of the present invention to cells and / or other immune organisms; preferably, the method is for non-therapeutic and diagnostic purposes.

[0042] Another aspect of the present invention provides a method for stimulating or activating B cells, characterized in that the method comprises administering one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more recombinant vaccines of the present invention to cells and / or other immune organisms; preferably, the method is for non-therapeutic and diagnostic purposes.

[0043] Another aspect of the present invention provides the use of one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more recombinant vaccines of the present invention in any of the following:

[0044] (a) preparing a drug for promoting the secretion of an antigen outside a cell in the form of exosomes;

[0045] (b) preparing a medicament for improving the humoral immunogenicity of an antigen;

[0046] (c) the preparation of a medicament for stimulating or activating B cells in an organism; or

[0047] (d) Preparation of medicines for treatment, vaccination or biological immunization.

[0048] Furthermore, it is characterized in that the drug is a drug for preventing and / or treating viral infectious diseases; preferably, the virus includes influenza virus, diarrhea virus, herpes virus, Epstein-Barr virus, hepatitis virus, or HPV virus; more preferably, the influenza virus is avian influenza virus, and the diarrhea virus is viral diarrhea virus; most preferably, the avian influenza virus is avian influenza H9N2 or H1N1, and the viral diarrhea virus is BVDV.

[0049] The fusion protein and recombinant vaccine of the present invention that can promote the secretion of cell membrane fixed protein have the following beneficial technical effects:

[0050] 1. The design idea of ​​the fusion molecular architecture described in the present invention is to express and secrete humoral immune antigens in the form of exosomes, and its key elements are the intracellular segment of low-affinity immunoglobulin gamma Fc region receptor II (FcγRII) and the WW domain of E3 ubiquitin protein ligase Nedd4 family member protein. The fusion molecular architecture of the present invention can localize the fusion protein to the cell membrane and bud (Budding) and secrete it to the extracellular space in the form of exosomes, promote the delivery of antigens to immune organs or germinal centers, effectively increase the probability of the humoral immune system recognizing antigen proteins, thereby improving the level of humoral immune response, and provide new technologies for the preparation of immune drugs that efficiently induce humoral immunity.

[0051] 2. In the fusion molecule architecture described in the present invention, the intracellular segment of the FcγRII protein is added between the antigen and the WW domain. The intracellular segment of FcγRII promotes the unidirectional transport of the formed vesicles between the endoplasmic reticulum and the Golgi apparatus, effectively improving the budding rate and secretion rate of the vesicles. The fusion molecule based on the present invention can greatly increase the probability of B cells recognizing antigens on the basis of maintaining the stability of the antigen protein conformation, thereby significantly enhancing the immunogenicity of the antigen target and improving the level of humoral immune response. The fusion molecule architecture of the present invention can effectively improve the expression efficiency of secretory proteins and membrane proteins, and promote the antigen proteins expressed by them to be presented on the surface of exosomes and secreted outside the cells, thereby improving the level of humoral immune response.

[0052] 3. The present invention can be used as a universal humoral immune framework for recombinant vaccines, which can stabilize the conformation of the antigen while allowing the antigen to be secreted outside the cell in the form of exosomes, effectively improving the humoral immunogenicity of the antigen and significantly activating the humoral immune response, thereby inducing the immune individual to produce higher levels of antigen-specific antibodies and memory B cells.

[0053] 4. Example 4 of the present invention shows that vaccine A is expressed both intracellularly and in the supernatant, proving that the WWdomain in the present invention has the ability to promote antigen secretion, but the expression level and secretion efficiency are low; vaccine B and vaccine C were both detected to be significantly expressed in the supernatant, proving that the combination of the FcγRII element and the WW domain element in the present invention can significantly improve the protein secretion effect. In addition, the FcγRII element in the present invention can be derived from different species, including but not limited to mouse, human, etc., and has similar functions.

[0054] 5. Based on the humoral immune vaccine of the present invention, in the in vitro expression experiment, the expression level of the antigen protein can be increased, and a large number of exosomes containing the antigen protein can be detected in the supernatant, achieving the expected technical effect. Example 5 of the present invention shows that the cells transfected with the vaccine E of the present invention detected significant expression of the target protein in the supernatant, which was significantly higher than the expression level in the cells, proving that the framework vaccine of the present invention can improve the expression and secretion efficiency of secretory antigens, which is better than the conventional secretory framework.

[0055] 6. Example 6 of the present invention shows that the diameter of the exosomes induced by the vaccine B of the present invention is between 35-150 nm, which is consistent with the characteristics of exosomes, and the target protein is distributed in clusters on the surface of the exosomes.

[0056] 7. The humoral immune vaccine based on the present invention can efficiently activate the humoral immune response in in vivo immune tests, stimulate the body to produce high levels of antigen-specific antibodies, and produce effective humoral immune protection, which is significantly better than conventional fusion molecular architectures.

[0057] 8. Example 7 of the present invention shows that the mRNA vaccine (vaccine F) with a conventional membrane protein framework only induced 6 mice (66.6%) to produce a high level of neutralizing antibodies, and it was not possible to ensure that all individuals produced effective humoral immunity. However, the recombinant nucleic acid vaccine (vaccine G) based on the present invention can induce all individuals (100%) to produce high levels of neutralizing antibodies, proving that the mRNA vaccine based on the present invention can effectively improve the humoral immune effect of the mRNA vaccine. The reason for the above results is that the framework of the present invention can promote the secretion of antigens outside the cells in the form of exosomes, increase the probability of antigen delivery to immune organs or germinal centers, thereby improving the efficiency of B cells recognizing antigens, and ultimately inducing a high level of humoral immunity.

[0058] 9. Example 8 of the present invention shows that the mRNA vaccine (vaccine G) based on the present invention can induce laying hens to produce neutralizing antibodies with an HI titer of 2 8 -2 10 , which is superior to the antibody level induced by the mRNA vaccine (Vaccine H) with the conventional secretory vaccine framework (2 3 -2 5 ), proving that the mRNA vaccine based on the present invention can not only improve the secretion effect of protein in in vitro cell experiments (such as Example 5), but also effectively improve the humoral immune effect of mRNA vaccines. The vaccine based on the present invention is not limited by the species of the immunized individuals, and can play a good role in inducing humoral immunity in both mice and laying hens.

[0059] 10. Example 9 of the present invention shows that in a single immunization experiment, the conventional mRNA vaccine (Vaccine I) can induce mice to produce neutralizing antibodies with an HI titer of 23 -2 4 The recombinant nucleic acid vaccine (vaccine J) based on the present invention induced mice to produce higher levels of neutralizing antibodies, with an HI titer of 2 4 -2 5 . It is proved that the mRNA vaccine based on the present invention can induce individuals to produce a higher level of humoral immune response in a short period of time after a single immunization.

[0060] 11. The humoral immune vaccine based on the present invention is applicable to a variety of antigens, and compared with conventional fusion molecular architectures, it can induce animal individuals to produce high levels of neutralizing antibodies in a short period of time. The vaccine based on the present invention provides a new recombinant vaccine molecular architecture, which has a better ability to promote the expression and secretion of humoral immune antigens and improve the level of humoral immunity, providing new ideas and methods for the design of recombinant vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 It is a non-limiting schematic diagram of the framework and vaccine involved in the present invention that can promote the secretion of cell membrane fixed protein.

[0062] Figure 2 Schematic diagram of the gene expression cassette for expressing the fusion protein molecular structure of the present invention.

[0063] Figure 3 Schematic diagram of the plasmid expressing the molecular structure of the fusion protein of the present invention, wherein SP is a signal peptide.

[0064] Figure 4A-4E This is a peak diagram of the quality control of the recombinant nucleic acid of the present invention and other fusion molecular structures, wherein Figure 4A This is the peak diagram of recombinant nucleic acid quality control based on WW domain. Figure 4B This is a quality control peak diagram of the recombinant nucleic acid I according to the present invention. Figure 4C This is a quality control peak diagram of the recombinant nucleic acid II according to the present invention. Figure 4D This is a peak diagram for quality control of recombinant nucleic acids based on conventional secretion architecture. Figure 4E This is a quality control peak diagram of the recombinant nucleic acid III based on the present invention.

[0065] Figure 5 These are the results of WB (Western blot) detection of in vitro expression of vaccines A, B, and C in HEK293 cells transfected with the vaccines.

[0066] Figure 6 This is the WB (Western blot) detection result of the in vitro expression of vaccine D in HEK293 cells transfected with the vaccine.

[0067] Figure 7This is the WB (Western blot) detection result of the in vitro expression of vaccine E in HEK293 cells transfected with the vaccine.

[0068] Figure 8 This is an electron micrograph of exosomes induced by the vaccine B of the present invention.

[0069] Fig. 9 Schematic diagram of the structure of vaccine F.

[0070] Fig.10 Schematic diagram of the structure of vaccine G.

[0071] Fig.11 This is a schematic diagram of the process of using vaccines F and G for mouse immunization and sampling.

[0072] Fig.12 These are the results of the hemagglutination inhibition titer test of H9N2 HA protein neutralizing antibodies induced by vaccines F and G in mice.

[0073] Fig.13 Schematic diagram of the structure of vaccine H.

[0074] Fig.14 This is a schematic diagram of the immunization and sampling process for laying hens using vaccines G and H.

[0075] Fig.15 These are the results of the hemagglutination inhibition titer test of neutralizing antibodies against H9N2 HA protein induced by vaccines G and H in laying hens.

[0076] Fig.16 Schematic diagram of the structure of vaccine I.

[0077] Fig.17 Schematic diagram of the structure of vaccine J.

[0078] Fig.18 Schematic diagram of the process of using vaccine I and vaccine J for mouse immunization and sampling.

[0079] Fig.19 The results of the hemagglutination inhibition test of H1N1 HA protein neutralizing antibodies induced by vaccine I and vaccine J in mice. DETAILED DESCRIPTION

[0080] Terms and Definitions

[0081] The term "FcγRII" refers to Low affinity immunoglobulin gamma Fc region receptorII. Specifically, "the intracellular segment of FcγRII" refers to the cytoplasmic region of the protein Low affinity immunoglobulin gamma Fc regionreceptor II, which can be the full length or partial fragment of the cytoplasmic region. "FcγRII" or "the intracellular segment of FcγRII" can be derived from any mammalian species, preferably human or mouse. FcγRII can be derived from mouse, and the amino acid sequence of the intracellular segment of mouse FcγRII is shown in SEQ ID NO: 3. FcγRII can be derived from guinea pig, and the amino acid sequence of the intracellular segment of guinea pig FcγRII is shown in SEQ ID NO: 4. FcγRII can be derived from human FcγRIIa, and the amino acid sequence of the intracellular segment of human FcγRIIa is shown in SEQ ID NO: 11.

[0082] The term "WW domain of E3 ubiquitin-protein ligase Nedd4 family member protein" refers to a sequence of E3 ubiquitin-protein ligase protein, Nedd4 family member protein, including WW1, WW2, WW3 and WW4. The source of the WW domain of Nedd4 is not limited to species, and the WW domain sequence of the corresponding species can be used for different species. Preferably, the WW domain of Nedd4 is derived from humans, and its amino acid sequence is shown in SEQ ID NO: 2.

[0083] The terms "antigen" or "immunogen" are used interchangeably and refer to a substance, usually a protein, that can induce an immune response in a subject. The term also refers to an immunologically active protein, i.e., once administered to a subject (directly or by administering a nucleotide sequence or vector encoding the protein to a subject), it can cause a humoral and / or cellular immune response against the protein. Preferably, the antigen refers to a vaccine antigen, i.e., a molecule that can induce a humoral immune response and / or a cellular immune response, resulting in the production of B and / or T lymphocytes specific for the antigen. The vaccine antigen can be a viral antigen, more preferably, the antigen can be a viral spike protein (S protein), a membrane protein (M protein), an envelope protein (E protein), and a nucleocapsid protein (N protein), or an outer membrane protein and secretory protein of a bacterium. Most preferably, the antigen refers to the HA protein of avian influenza or the E2 protein of BVDV (bovine viral diarrhea virus).

[0084] The term "exosome" refers to membrane vesicles secreted by cells, which are approximately 30-160 nm in size and can be released into the extracellular space by fusion of multivesicular bodies with the cell membrane or by budding from the cell membrane.

[0085] The term "administration" or "vaccination" refers to administration based on the nucleic acid vaccine or vaccine composition of the present invention preferably via intramuscular or subcutaneous routes, although other routes of administration can also be used, for example, oral, intranasal (e.g., aerosol or other non-injection administration), intralymph node, intradermal, intraperitoneal, rectal or vaginal administration, or by a combination of routes. Administration in the neck muscle of an animal is preferred. Boosting regimens can be used to adjust the dosage regimen to provide optimal immunity.

[0086] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0087] The term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide", "recombinant protein", "fusion protein", etc.

[0088] The term "recombinant expression vector" refers to a DNA structure used to express a polynucleotide encoding a desired polypeptide, for example. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence that is transcribed into mRNA and translated into protein; and (3) appropriate transcription and translation start and stop sequences. The recombinant expression vector is constructed in any suitable manner and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from a combination of plasmids and phage DNA, DNA from viruses such as lentivirus, retrovirus, vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. Both self-replicating and non-self-replicating vectors are included.

[0089] The term "mRNA" refers to messenger RNA, which is translated into Chinese as "messenger ribonucleic acid". It is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template, carries genetic information and can guide protein synthesis.

[0090] The term "5'-UTR", which refers to the "5' untranslated region" or "5'UTR", is a portion of a gene that is transcribed into a primary RNA transcript (pre-mRNA) and is located upstream of the coding sequence. The primary transcript is the initial RNA product, containing introns and exons, produced by transcription from DNA. Many primary transcripts must undergo RNA processing to form physiologically active RNA. The processing to form mature mRNA includes modifying the ends, removing introns, capping and / or shearing off individual rRNA molecules from the precursor RNA. Therefore, the 5'UTR of an mRNA is a portion of the mRNA that is not translated into protein and is located upstream of the coding sequence. In a genomic sequence, the 5'UTR is usually defined as the region located between the transcription start site and the start codon. The 5' untranslated region (5'UTR) of vertebrate mRNA can be tens to hundreds of bases in length.

[0091] The term "3'-UTR", meaning "3'-untranslated region" or "3'UTR", refers to a region located at the 3' end of a gene, downstream of the stop codon of the protein coding region, and which is transcribed but not translated into an amino acid sequence, or to the corresponding region in an RNA molecule. The 3'-untranslated region typically extends from the stop codon of the translation product to a poly(A) sequence that is typically attached after the transcription process. The 3'-untranslated region of mammalian mRNA typically has a homology region known as the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-untranslated region may contain one or more inverted repeats and may fold to produce a stem-loop structure that acts as a barrier to exoribonucleases or interacts with proteins (e.g., RNA binding proteins) that are known to increase RNA stability.

[0092] The term "polyA", meaning "polyadenylic acid sequence", "poly (A) sequence" or "poly (A) tail", refers to a sequence of adenylic acid residues usually located at the 3' end of an RNA molecule. The present invention allows such a sequence to be attached during RNA transcription by a DNA template based on repeated thymidylic acid residues in a strand complementary to the coding strand, however, the sequence is not normally encoded in DNA, but is attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription in the nucleus.

[0093] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce recombinant vaccines based on the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells.

[0094] The terms "individual", "patient" or "subject" include animals. Animals include, but are not limited to, domestic animals (e.g., pigs, cows, sheep, cats, dogs, horses, chickens and ducks, etc.), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., rabbits, mice and rats, etc.).

[0095] The terms "transformation, transfection, and transduction" have the meanings generally understood by those skilled in the art, namely, the process of introducing exogenous DNA or RNA into a host.

[0096] The term "pharmaceutical composition" or "drug combination" refers to auxiliary materials widely used in the field of drug production. The main purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature and / or has specific functionality, and also to provide a method so that it can be effectively absorbed in the body of a subject. A pharmaceutically acceptable carrier can be an inert filler or an effective component that provides a certain function for the pharmaceutical combination (such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, adhesives, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, glidants, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0097] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0098] The present invention discloses a fusion molecule framework that promotes the secretion of a target protein into the extracellular space in the form of exosomes, a method for preparing a recombinant vaccine based on the framework, and its application. A person skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve it. It should be noted in particular that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0099] The present invention is further described in detail below in conjunction with the accompanying drawings and implementation cases. Among them, as a preferred embodiment, a nucleic acid vaccine framework is selected for the preparation of a recombinant vaccine, but the protection scope of the present invention is not limited to the above content; the reagents, instruments, experimental animals, etc. used in the embodiments of the present invention are all commercially available in the field or can be obtained through conventional channels; the experimental operating procedures, steps, parameters and other methods and operations mainly used in the embodiments are all well known and can be implemented by ordinary biotechnical personnel in the field.

[0100] Example 1 Construction of the recombinant nucleic acid vaccine of the present invention

[0101] Figure 1 The following is a non-limiting schematic diagram of the framework and vaccine involved in the present invention that can promote the secretion of cell membrane fixed proteins. Figure 1 The recombinant nucleic acid vaccine shown, exemplarily, first constructs a gene expression cassette, the encoding amino acid sequence of which is shown in SEQ ID NO: 1, the full length of the sequence is 814 amino acids, and the specific composition is from the N-terminus to the C-terminus, the 1st to 560th amino acids are the full length of the HA antigen, and the full length of the HA antigen specifically includes the HA signal peptide (amino acids 1-18), HA target antigen (amino acids 19-524), HA transmembrane region (amino acids 525-549); Linker (Linker 1, amino acids 561-566); guinea pig FcγRII intracellular segment (amino acids 567-6116); Linker (Linker 2, amino acids 612-615); WW domain (amino acids 616-801); Linker (Linker 3, amino acids 802-808); His tag (amino acids 809-814), which can be used for WB detection. The schematic diagram of the gene expression cassette expressing the fusion protein molecular architecture of the present invention is shown in Figure 2As shown, the expression cassette includes: 5'UTR, CDS region, 3'UTR, PolyA from 5' to 3' end, wherein the elements included in the CDS region are: signal peptide, target antigen, transmembrane region, FcγRII intracellular segment, WW domain, wherein the full length of the antigen can include a signal peptide, a target antigen, and a transmembrane region, and the sources thereof are the same as a whole antigen. It can also be that the target antigen and the signal peptide and transmembrane region are from different sources and are combined. A linker sequence can be optionally inserted between each element, and the linker sequence can be a GS linker sequence. Preferably, the WW domain is the WW domain of the E3 ubiquitin protein ligase Nedd4 family member protein located in the membrane, and its amino acid sequence is shown in SEQ ID NO: 2. The intracellular segment of FcγRII can be derived from mice, and its amino acid sequence is shown in SEQ ID NO: 3, or from guinea pigs, and its amino acid sequence is shown in SEQ ID NO: 4. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained by gene synthesis (commissioned to be synthesized by GenScript). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector that can be used for in vitro RNA transcription to obtain a vector plasmid for preparing a recombinant nucleic acid vaccine (the plasmid schematic diagram is shown in FIG. Figure 3 as shown).

[0102] According to the above method, vectors (1) to (5) were prepared for comparative experiments. The five vectors involved two groups of comparative experiments, in which vectors (1) to (3) were one group and vectors (4) to (5) were another group.

[0103] In order to more rigorously demonstrate that the present invention has the effect of promoting antigen secretion and prove that the present invention is applicable to different types of antigens, in this embodiment, the vectors of the same group of comparative experiments use the same antigen, and the sequence optimization method remains consistent. In this embodiment, illustratively, target antigen 1 is the full-length HA protein of H9N2 (the N-terminus contains a signal peptide, the C-terminus contains a transmembrane region, and its amino acid is shown in SEQ ID NO: 5), which is a membrane protein; target antigen 2 is the E2 antigen protein of BVDV (see the published patent CN116284272B, the announcement date is 2023-08-25, its amino acid is shown in SEQ ID NO: 6, and does not contain its own signal peptide and transmembrane region), which is a secreted protein.

[0104] (1) Preparation vector of recombinant nucleic acid vaccine (vaccine A) based on WW domain

[0105] Step a: Synthesize the "target antigen 1 (N-terminal contains a signal peptide, C-terminal contains a transmembrane region)-WW domain" gene fragment. The amino acids of target antigen 1 are shown in SEQ ID NO: 5, the amino acids of the signal peptide in target antigen 1 are shown in SEQ ID NO: 12, the amino acids of the transmembrane region in target antigen 1 are shown in SEQ ID NO: 13, and the amino acids of WW domain are shown in SEQ ID NO: 2.

[0106] Step b: Construct a nucleic acid vaccine framework vector.

[0107] The nucleic acid vaccine framework vector comprises a 5'-UTR and a 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing a DNA vaccine.

[0108] Step c: preparing recombinant plasmid.

[0109] The gene synthesized in step a is inserted into the vector structure of step b to obtain a recombinant nucleic acid vaccine preparation vector based on the WW domain.

[0110] (2) Preparation of vector I based on the recombinant nucleic acid vaccine (vaccine B) of the present invention

[0111] Step a: As in step a of (1) above, synthesize the gene fragment of "target antigen 1 (N-terminus contains signal peptide, C-terminus contains transmembrane region)-mouse FcγRII intracellular segment-WW domain". Wherein, the amino acids of target antigen 1 are shown in SEQ ID NO: 5, the amino acids of the signal peptide in target antigen 1 are shown in SEQ ID NO: 12, the amino acids of the transmembrane region in target antigen 1 are shown in SEQ ID NO: 13, the amino acids of the mouse FcγRII intracellular segment are shown in SEQ ID NO: 3, and the amino acids of the WW domain are shown in SEQ ID NO: 2.

[0112] Step b: As described in step b of (1) above, construct a nucleic acid vaccine framework vector.

[0113] Step c: preparing recombinant plasmid.

[0114] The gene synthesized in step a is inserted into the vector structure of step b to obtain a preparation vector for expressing the recombinant nucleic acid vaccine I based on the present invention.

[0115] (3) Preparation of vector II based on the recombinant nucleic acid vaccine (vaccine C) of the present invention

[0116] Step a: As in step a of (1) above, synthesize the gene fragment of "target antigen 1 (N-terminus contains signal peptide, C-terminus contains transmembrane region)-guinea pig FcγRII intracellular segment-WW domain". The amino acids of target antigen 1 are shown in SEQ ID NO: 5, the amino acids of the signal peptide in target antigen 1 are shown in SEQ ID NO: 12, the amino acids of the transmembrane region in target antigen 1 are shown in SEQ ID NO: 13, the amino acids of the guinea pig FcγRII intracellular segment are shown in SEQ ID NO: 4, and the amino acids of WW domain are shown in SEQ ID NO: 2.

[0117] Step b: As described in step b of (1) above, construct a nucleic acid vaccine framework vector.

[0118] Step c: preparing recombinant plasmid.

[0119] The gene synthesized in step a is inserted into the vector structure of step b to obtain vector II for expressing the recombinant nucleic acid vaccine preparation based on the present invention.

[0120] (4) Preparation vector of recombinant nucleic acid vaccine (Vaccine D) based on conventional secretion architecture

[0121] Step a: As in step a of (1) above, synthesize the "signal peptide-target antigen 2" gene fragment. The signal peptide is derived from the Fc domain of a bovine antibody protein, and its amino acids are shown in SEQ ID NO: 14; the amino acids of target antigen 2 are shown in SEQ ID NO: 6.

[0122] Step b: As described in step b of (1) above, construct a nucleic acid vaccine framework vector.

[0123] Step c: preparing recombinant plasmid.

[0124] The gene synthesized in step a is inserted into the vector structure of step b to obtain a recombinant nucleic acid vaccine preparation vector expressing a conventional secretion structure.

[0125] (5) Preparation of vector III based on the recombinant nucleic acid vaccine (vaccine E) of the present invention

[0126] Step a: As in step a of (1) above, synthesize the gene fragment of "signal peptide-target antigen 2-transmembrane region-guinea pig FcγRII intracellular segment-WW domain". The signal peptide is derived from the Fc domain of bovine antibody protein, and its amino acid sequence is shown in SEQ ID NO: 14. The amino acid sequence of target antigen 2 is shown in SEQ ID NO: 6. The transmembrane region is derived from bovine CD80, and its amino acid sequence is shown in SEQ ID NO: 15. The amino acid sequence of guinea pig FcγRII intracellular segment is shown in SEQ ID NO: 4. The amino acid sequence of WW domain is shown in SEQ ID NO: 2.

[0127] Step b: As described in step b of (1) above, construct a nucleic acid vaccine framework vector.

[0128] Step c: preparing recombinant plasmid.

[0129] The gene synthesized in step a is inserted into the vector structure of step b to obtain vector III for expressing the recombinant nucleic acid vaccine preparation based on the present invention.

[0130] Table 1 Protein amino acid sequences of the structural elements involved in the present invention

[0131] Amino acid sequence and sequence number Gene expression cassette encoding protein (SEQ ID NO: 1) WW domain APLPPGWEQRVDQHGRVYYVDHVEKRTTWDRPEPLPPGWERRVDNMGRIYYVDHFTRTTTWQRPTLESVRNYEQWQLQRSQLQGAMQQFNQRFIYGNQDLFATSQSKEFDPLGPLPPGWEKRTDSNGRVYFVNHNTRITQWEDPRSQGQLNEKPLPEGWEMRFTVDGIPYFVDHNRRTTTYIDPRT (SEQ ID NO:2) Intracellular domain of murine FcγRII ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPY (SEQ ID NO:3) Intracellular domain of guinea pig FcγRII GNPEHREMGETLPEDPGEYSVVFGGSMMSCPGLPDGLEPARTDLS (SEQ ID NO:4) HA protein of H9N2 METISLMTILLIAAASNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTCTIEGLIYGNPACDLLLEGRGWSYIVERSSAVNGTCYPGNVENLEELRTIFSSASSYQRIQIFPDTTWNVSYTGTSMSCSDSFYRSMRWLTQKSGFYPVQDAQYTNNRGKNILFVWGIHHPPTYSAQTTLYIRNDTTTIVTTEELNRTFKPVIGPRPIVNGQQGRIDYYWSVLKPGQTLRVRTNGNLIAPWYGHVLSGGSHGRILKTDLKSGNCLVQCQTEKGGLNSTLPFHNISKYAFGNCPKYVKVNSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRESTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI (SEQ ID NO:5) E2 protein of BVDV HLDCKPEFSYAIAKSDRIGPLGAEGLTTTWKDYSHEMKLEDTMVIAWCKDGKFVYLQRCTRETRYLAILHSRALPTSVVFKKLFDGRRQEDTVEMDDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCMLANRDTLDTAVVRTYRRSKPFPYRQGCGGSGGGGSGGLPDCKPDFSYAIAKNDEIGPLGATGLTTQWYEYSDGMRLQDTEVVVWCKDGEFKYLIRCEREARYLAILHTRALPTSVVFEKILNGKEQEDVVEMDDNFEFGLCPCDAKPLVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTLANKDTLATTVVRTYKRHRPFPYRQGCGGSGGGGSGGFPECKEGFQYAISKDKKMGLLGPESLTTTWHLPTKKIVDSMVQVWCEGKDLKILRTCTKEERYLVAVHERALSTSAEFLQISDGTTGPEVIDMPDDFEFGLCPCDSKPVIKGKFNTSLLNGPAFQMVCPQGWTGTIECILANQDTLDTTVIRTYRRTTPFQRRKWC (SEQ ID NO:6) HA protein of H1N1 (SEQ ID NO:7) Linker Sequence 1 GSGSGS (SEQ ID NO:8) Linker Sequence 2 GGGS (SEQ ID NO:9) Linker Sequence 3 GSGSGSG (SEQ ID NO: 10) Human FcγRIIa intracellular domain CRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNSNN (SEQ ID NO:11) Signal peptide of H9N2 HA protein METISLMTILLIAAASNA (SEQ ID NO:12) Transmembrane region of H9N2 HA protein LTIYSTVASSLVIAMGFAAFLFWAM (SEQ ID NO:13) Signal peptide of bovine antibody protein Fc domain MNPLWTLLFVLSAPRGVLS (SEQ ID NO:14) Transmembrane region of bovine CD80 protein LTWTIIIPVSAFGISVIIAVILTCLTC (SEQ ID NO:15)

[0132] Example 2 Preparation of the recombinant nucleic acid vaccine of the present invention

[0133] (1) Preparation of capped mRNA vaccines

[0134] Step a: The vector plasmid used to produce the capped mRNA vaccine in Example 1 is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0135] Step b: The linearized plasmid is subjected to an in vitro co-transcription capping reaction to add a 7-methylated guanylate cap structure to the 5' end of the transcribed mRNA, and the template DNA is degraded.

[0136] (2) Preparation of non-capped mRNA vaccines

[0137] Step a: The vector plasmid used to produce the non-capped mRNA vaccine in Example 1 is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0138] Step b: The linearized plasmid is subjected to an in vitro non-capped transcription reaction and the template DNA is degraded.

[0139] (3) DNA vaccine preparation

[0140] Step a: Amplify the vector plasmid used to produce the DNA vaccine in Example 1 to obtain a large amount of target plasmid for purification.

[0141] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0142] Example 3 Quality Control of In Vitro Transcription of Recombinant Nucleic Acid and Vaccine Preparation of the Present Invention

[0143] Vaccine A (recombinant nucleic acid based on WW domain), vaccine B (recombinant nucleic acid I of the present invention), vaccine C (recombinant nucleic acid II of the present invention), vaccine D (recombinant nucleic acid based on conventional secretion framework), and vaccine E (recombinant nucleic acid III of the present invention) were prepared by the method for preparing capped mRNA vaccines in Example 2. The purity of the produced recombinant nucleic acids was tested, and the purity of the 5 groups of recombinant nucleic acids used for comparative experiments was greater than 80%. The quality control peaks of the recombinant nucleic acids based on the present invention and other fusion molecular frameworks are shown in the figure. Figure 4A , Figure 4B , Figure 4C , Figure 4D , Figure 4E As shown. Specifically described as follows: (1) Recombinant nucleic acid based on WW domain (vaccine A), purity is 80.9%; (2) Recombinant nucleic acid I based on the present invention (vaccine B), purity is 80.1%; (3) Recombinant nucleic acid II based on the present invention (vaccine C), purity is 80.3%; (4) Recombinant nucleic acid based on conventional secretion structure (vaccine D), purity is 92.5%; (5) Recombinant nucleic acid III based on the present invention (vaccine E), purity is 83.1%. The above purities all meet the quality requirements of cell transfection experiments and vaccine production.

[0144] Example 4 Promoting the secretory expression of membrane protein antigens based on the recombinant nucleic acid of the present invention

[0145] Vaccines A, B, and C in Example 3 were transfected into HEK293T cells using a cell transfection reagent. After 48 hours of in vitro culture, the supernatant was collected and concentrated to obtain extracellular components, and the cells were lysed to obtain cell membrane and intracellular components, both of which were detected by Western blot.

[0146] Figure 5 The results of WB (Western blot) detection of in vitro expression of vaccines A, B, and C in HEK293 cells were as follows: Figure 5 As shown, after transfection of all vaccines, the target protein was detected in the supernatant, and the protein molecular weights of vaccines A, B, and C are shown in Table 2. Among them, vaccine A was expressed both intracellularly and in the supernatant, proving that the WW domain in the present invention has the ability to promote antigen secretion, but the expression amount and secretion efficiency are low; vaccine B and vaccine C were both detected to be significantly expressed in the supernatant, proving that the combination of the FcγRII element and the WW domain element in the present invention can significantly improve the protein secretion effect. In addition, the FcγRII element in the present invention can be derived from different species, including but not limited to mouse, human, etc., and has similar functions. The experimental results are consistent with the theory and expectations.

[0147] Since the target antigen 1 used in this example is the HA protein of H9N2, which is a membrane protein, it will not be secreted into the extracellular supernatant during conventional expression. Therefore, if the target protein is detected in the supernatant of the sample in this example, it is sufficient to prove that the fused exosome-related elements have played a role.

[0148] Table 2 Protein molecular weight of vaccines A, B, and C

[0149] name describe Protein molecular weight (kDa) Vaccine A Recombinant nucleic acids based on WW domain 88.6 Vaccine B Recombinant nucleic acid I based on the present invention 91.8 Vaccine C Recombinant nucleic acid II based on the present invention 91.7

[0150] Example 5 Promoting secretory expression of secretory antigens based on the recombinant nucleic acid of the present invention

[0151] Vaccines D and E in Example 3 were transfected into HEK293T cells using a cell transfection reagent, and the supernatant was collected and concentrated after 48 hours of in vitro culture to obtain extracellular components, and the cells were lysed to obtain cell membrane and intracellular components, both of which were detected by Western blot. The protein molecular weights of vaccines D and E are shown in Table 3.

[0152] Table 3 Protein molecular weight of vaccines D and E

[0153] name describe Protein molecular weight (kDa) Vaccine D Recombinant nucleic acids based on conventional secretion architectures 90.2 Vaccine E Recombinant nucleic acid III based on the present invention 124.3

[0154] Figure 6 The results of WB (Western blot) detection of in vitro expression of vaccine D in HEK293 cells are shown in Figure 2. Figure 6 As shown, in cells transfected with vaccine D, the expression of the target protein was detected both intracellularly and in the supernatant, and the intracellular expression level was significantly higher than that in the supernatant, indicating that the conventional secretion structure can secrete the secretory protein (target antigen 2) outside the cell, but the efficiency is low. Figure 7 The results of WB (Western blot) detection of in vitro expression of vaccine E in HEK293 cells were as follows: Figure 7 As shown, in the cells transfected with the vaccine E of the present invention, significant expression of the target protein was detected in the supernatant, which was significantly higher than the expression level in the cells, proving that the present invention can improve the expression and secretion efficiency of secretory antigens, which is superior to conventional secretion structures.

[0155] Example 6 Vaccines of the present invention promote secretion of antigens in the form of exosomes

[0156] The vaccine B in Example 3 was transfected into HEK293T cells using a cell transfection reagent. After 48 hours of in vitro culture, the supernatant was collected and concentrated by low-temperature centrifugation through an ultrafiltration tube to obtain exosome components.

[0157] The collected exosome samples were imaged by cryo-electron microscopy, and the morphology of the exosomes induced by the vaccine B of the present invention was as follows Figure 8As shown, the diameter is between 35-150 nm, which is consistent with the characteristics of exosomes, and the target protein is distributed in clusters on the surface of exosomes, which is consistent with Figure 1 The non-limiting schematic diagram shown Figure 1 The results are consistent with those of the present invention, proving that the vaccine according to the present invention can play the above-mentioned function, which is consistent with the expectation.

[0158] therefore, Figure 5 and Figure 7 The protein expression levels in the concentrated supernatant shown in the figure are all antigen proteins secreted outside the cells in the form of exosomes, which is in line with the technical principles and expected effects.

[0159] Example 7: Improvement of humoral immunity in mice by the H9N2 recombinant nucleic acid vaccine of the present invention

[0160] In order to demonstrate the effectiveness of the present invention in animals, this example constructs vaccines F and G containing H9N2 humoral immune antigens based on the conventional membrane protein vaccine architecture and the vaccine architecture of the present invention, respectively. The full-length sequence of antigen H9N2 HA is shown in SEQ ID NO: 5, which is used for the mouse immunization experiment of this example. The antigen sequences of the two vaccines are the same, and there is only a difference in the architecture.

[0161] The schematic diagram of the vaccine F structure is as follows Fig. 9 As shown, from 5' to 3' end, it includes T7 promoter, 5'UTR, H9N2 HA full-length sequence, H9N2 HA full-length sequence (amino acid sequence is shown in SEQ ID NO: 5, which includes its own signal peptide and transmembrane region) signal peptide as shown in SEQ ID NO: 12, transmembrane region as shown in SEQ ID NO: 13, 3'UTR, the specific sequence of each element is shown in Table 1.

[0162] The schematic diagram of the vaccine G structure is as follows Fig.10 As shown, from 5' to 3' end, it includes T7 promoter, 5'UTR, H9N2 HA full-length sequence, H9N2 HA full-length sequence (amino acid sequence is shown in SEQ ID NO:5, which includes its own signal peptide and transmembrane region) signal peptide as shown in SEQ ID NO:12, transmembrane region as shown in SEQ ID NO:13, Linker sequence (which is Linker sequence 1, amino acid sequence as shown in SEQ ID NO:8), FcγRII intracellular segment (amino acid sequence as shown in SEQ ID NO:4), Linker sequence (which is Linker sequence 2, amino acid sequence as shown in SEQ ID NO:9), WW domain (amino acid sequence as shown in SEQ ID NO:2), 3'UTR, the specific sequence of each element is shown in Table 1.

[0163] A total of 18 female Balb / c mice aged 6-8 weeks were selected for the experiment. After 3-7 days of adaptive feeding, they were randomly divided into 2 groups according to body weight. There were 9 mice in each group, for a total of 2 groups. As shown in Table 4, the dosage in this table and below refers to the amount of active ingredient.

[0164] Table 4 Grouping and treatment of animals in the immunization experiment of Example 7

[0165]

[0166] The mice in the above groups were immunized once and twice according to the immunization process in Table 4. Blood was collected on day 0 (before the first immunization) and day 28 (Day 28) to measure the hemagglutination inhibition titer (HI titers). The serum extracted from the three mice in each group was mixed into one biological sample, so three serum mixed biological samples were obtained for detection after sampling in each group. The immunization and sampling process is as follows Fig.11 shown.

[0167] The results of the hemagglutination inhibition titer test of H9N2 HA protein neutralizing antibodies induced by vaccines F and G in mice are as follows Fig.12 As shown in the figure, the experimental results show that the mRNA vaccine (vaccine F) with a conventional membrane protein structure only induced 6 mice (66.6%) to produce a high level of neutralizing antibodies, which cannot ensure that all individuals produce effective humoral immunity. However, the recombinant nucleic acid vaccine (vaccine G) based on the present invention can induce all individuals (100%) to produce high levels of neutralizing antibodies, proving that the mRNA vaccine based on the present invention can effectively improve the humoral immunity effect of the mRNA vaccine.

[0168] The reason for the above results is that the present invention can promote the secretion of antigens outside cells in the form of exosomes, increasing the probability of antigen delivery to immune organs or germinal centers, thereby improving the efficiency of B cells recognizing antigens and ultimately inducing a high level of humoral immunity.

[0169] Example 8: Improving the humoral immunity of laying hens based on the H9N2 recombinant nucleic acid vaccine of the present invention

[0170] In order to demonstrate the effectiveness of the vaccine based on the present invention in different animals and compare the differences between the present invention and conventional secretion frameworks, this example continued to use the humoral immune antigens in Example 7, adopted the conventional secretion vaccine framework, and newly constructed vaccine H, and conducted a comparative experiment with the vaccine G based on the present invention in Example 7. This example conducted an immunization experiment in laying hens. The antigen sequences of the two vaccines were the same, and both were vaccine frameworks that promoted protein secretion. Vaccine H was a conventional secretion framework element, and vaccine G was a framework element based on the present invention.

[0171] The schematic diagram of the vaccine H structure is as follows Fig.13As shown, from 5' to 3' end, it includes T7 promoter, 5'UTR, H9N2 HA extracellular domain (which includes its own signal peptide but does not include its own transmembrane region), the signal peptide in the H9N2 HA extracellular domain is shown in SEQ ID NO: 12, 3'UTR, and the specific sequences of each element are shown in Table 1.

[0172] A total of 8 SPF laying hens were selected for the experiment and divided into 2 groups, with 4 hens in each group. As shown in Table 5, the dosage in this table and below refers to the amount of active ingredient.

[0173] Table 5 Grouping and treatment of animals in the immunization experiment of Example 8

[0174]

[0175] The above-mentioned groups of laying hens were immunized once and twice according to the immunization process in Table 5. Blood was collected on day 0 (before the first immunization), day 28 (Day 28), and day 35 (Day 35) to measure the hemagglutination inhibition titer (HI titers). The serum of each laying hen was tested separately. The immunization and sampling process is as follows Fig.14 shown.

[0176] The results of the hemagglutination inhibition titer test of H9N2 HA protein neutralizing antibodies induced by vaccine G and vaccine H in laying hens are as follows Fig.15 As shown in the experimental results, it can be seen that the mRNA vaccine (vaccine G) based on the present invention can induce laying hens to produce neutralizing antibodies with an HI titer of 2 8 -2 10 , which is superior to the antibody level induced by the mRNA vaccine (Vaccine H) with the conventional secretory vaccine framework (2 3 -2 5 ). It is proved that the mRNA vaccine based on the present invention can not only improve the secretion effect of protein in in vitro cell experiments (such as Example 5), but also effectively improve the humoral immune effect of mRNA vaccine.

[0177] In addition, the vaccine based on the present invention is not limited by the species of the immunized individual and can exert a good effect of inducing humoral immunity in both mice and laying hens.

[0178] Example 9: The H1N1 recombinant nucleic acid vaccine of the present invention induces mice to produce high levels of antibodies in a short period of time

[0179] In order to demonstrate that the present invention is not limited by the target and can induce a high level of humoral immune response in an individual in a short period of time after a single immunization, this example constructs Vaccine I and Vaccine J containing H1N1 humoral immune antigens based on the conventional vaccine architecture and the vaccine architecture of the present invention, respectively. The antigen sequence is shown in SEQ ID NO: 7, which is used for the mouse immunization experiment of this example. The antigen sequences of the two vaccines are the same, and there is only a difference in the architecture.

[0180] The schematic diagram of the vaccine I is as follows: Fig.16 As shown, from 5' to 3' end, it includes T7 promoter, 5'UTR, H1N1 HA full-length sequence (including signal peptide and transmembrane region), 3'UTR, and the specific sequence of each element is shown in Table 1.

[0181] The schematic diagram of the vaccine J structure is as follows Fig.17 As shown, from 5' to 3' end, it includes T7 promoter, 5'UTR, H1N1 HA full-length sequence (including signal peptide and transmembrane region), Linker sequence, FcγRII intracellular segment, Linker sequence, WW domain, 3'UTR, and the specific sequence of each element is shown in Table 1.

[0182] Ten female Balb / c mice aged 6-8 weeks were selected for the experiment. After 3-7 days of adaptive feeding, they were randomly divided into two groups according to body weight. There were five mice in each group, for a total of two groups. As shown in Table 6, the dosage in this table and below refers to the amount of active ingredient.

[0183] Table 6 Grouping and treatment of animals in the immunization experiment of Example 9

[0184] Vaccine Group Immunization Schedule I Dose: 10 μg / mouse, injected intramuscularly in the left leg J Dose: 10 μg / mouse, injected intramuscularly in the left leg

[0185] The mice in the above groups were immunized once according to the immunization process in Table 6. Blood was collected on Day 14 to measure the hemagglutination inhibition titer (HI titers). The serum extracted from each mouse was tested separately. Fig.18 shown.

[0186] The results of the hemagglutination inhibition titer test of H1N1 HA protein neutralizing antibodies induced by vaccine I and vaccine J in mice are as follows Fig.19 As shown in the experimental results, the conventional mRNA vaccine (vaccine I) can induce mice to produce neutralizing antibodies with an HI titer of 2 3 -2 4 The recombinant nucleic acid vaccine (vaccine J) based on the present invention induced mice to produce higher levels of neutralizing antibodies, with an HI titer of 2 4 -2 5 . It is proved that the mRNA vaccine based on the present invention can induce individuals to produce a higher level of humoral immune response in a short period of time after a single immunization, which is in line with expectations.

[0187] In summary, the present invention provides a fusion molecular framework that promotes the secretion of proteins into the extracellular space in the form of exosomes, which can not only effectively improve the protein expression level and secretion efficiency, but also can be applied to the production and development of animal immune drugs, significantly improve the immunogenicity of humoral immune antigens, and induce vaccinated individuals to produce a high level of humoral immune response. The effect is better than the current existing technology, and it has extremely high commercial value and broad application prospects.

[0188] The above embodiments of the present disclosure are merely examples for clearly illustrating the present disclosure, and are not intended to limit the implementation methods of the present disclosure. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present disclosure shall be included in the scope of protection of the claims of the present disclosure.

Claims

1. A fusion protein, characterized in that From N-terminus to C-terminus, it includes signal peptide, target antigen, transmembrane region, FcγRII intracellular segment, and E3 ubiquitin protein ligase Nedd4 family member protein WW domain; among them, the transmembrane region, FcγRII intracellular segment, and E3 ubiquitin protein ligase Nedd4 family member protein WW domain are connected by a linker sequence.

2. The fusion protein according to claim 1, characterized in that The antigen is the complete sequence of a membrane protein or secretory protein of a pathogen or a partial sequence containing an immune epitope.

3. The fusion protein according to claim 2, characterized in that The membrane protein or secretory protein of the pathogen is the full-length sequence of the HA protein of avian influenza or the E2 protein of BVDV (bovine viral diarrhea virus).

4. The fusion protein according to claim 1, characterized in that The antigen is the HA protein of avian influenza H9N2, or the E2 protein of BVDV (bovine viral diarrhea virus), or the HA protein of avian influenza H1N1.

5. The fusion protein according to claim 4, characterized in that The amino acid sequence of the HA protein of avian influenza H9N2 is shown in SEQ ID NO:5, the amino acid sequence of the E2 protein of BVDV is shown in SEQ ID NO:6, and the amino acid sequence of the HA protein of avian influenza H1N1 is shown in SEQ ID NO:

7.

6. The fusion protein according to claim 1, characterized in that The transmembrane region is selected from the transmembrane motif of the antigen sequence, or is truncated from the transmembrane motif of other membrane proteins.

7. The fusion protein according to claim 6, characterized in that The transmembrane motif truncated from other membrane proteins is from CD80 protein of any species.

8. The fusion protein according to claim 6, characterized in that The amino acid sequence of the self-transmembrane motif is shown as SEQ ID NO:

13.

9. The fusion protein according to claim 7, characterized in that The amino acid sequence of the transmembrane motif of the CD80 protein is shown in SEQ ID NO:

15.

10. The fusion protein according to claim 1, characterized in that The intracellular segment of FcγRII is selected from the full-length sequence of the cytoplasmic region.

11. The fusion protein according to claim 10, characterized in that The intracellular segment of FcγRII is derived from mouse, guinea pig or human.

12. The fusion protein according to claim 11, characterized in that The amino acid sequence of the intracellular segment of FcγRII derived from mouse is shown in SEQ ID NO: 3, or the amino acid sequence of the intracellular segment of FcγRII derived from guinea pig is shown in SEQ ID NO: 4, or the intracellular segment of FcγRII is derived from human FcγRIIa, and the amino acid sequence of the intracellular segment of human FcγRIIa is shown in SEQ ID NO:

11.

13. The fusion protein according to claim 1, characterized in that The WW domain is located on the cytoplasmic side, and its amino acid sequence is shown in SEQ ID NO:

2.

14. The fusion protein according to claim 1, characterized in that The linker sequence is shown in SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:

10.

15. The fusion protein according to claim 1, characterized in that The fusion protein comprises the following elements from N-terminus to C-terminus: (1) the signal peptide; the amino acid sequence of the signal peptide is shown in SEQ ID NO: 12 or SEQ ID NO: 14; (2) The antigen; the antigen is the HA protein of avian influenza H9N2 with an amino acid sequence as shown in SEQ ID NO:5, or the E2 protein of BVDV with an amino acid sequence as shown in SEQ ID NO:6, or the HA protein of avian influenza H1N1 with an amino acid sequence as shown in SEQ ID NO:7; (3) the transmembrane region; the transmembrane region comprises a transmembrane motif, and the transmembrane motif is a transmembrane motif of the antigen sequence itself, or a transmembrane motif from the CD80 protein; the amino acid sequence of the transmembrane motif of the CD80 protein is shown in SEQ ID NO: 15; (4) the intracellular segment of FcγRII; the intracellular segment of FcγRII is the intracellular segment of FcγRII derived from mouse, whose amino acid sequence is shown in SEQ ID NO: 3, or the intracellular segment of FcγRII derived from guinea pig, whose amino acid sequence is shown in SEQ ID NO: 4; (5) the WW domain; the amino acid sequence of the WW domain is shown in SEQ ID NO: 2; Among them, the transmembrane region, FcγRII intracellular segment, and WW domain are connected by a linker sequence.

16. The fusion protein according to claim 1, characterized in that The fusion protein comprises the following elements from N-terminus to C-terminus: a signal peptide with an amino acid sequence as shown in SEQ ID NO: 14, a target antigen 2 with an amino acid sequence as shown in SEQ ID NO: 6, a transmembrane region with an amino acid sequence as shown in SEQ ID NO: 15, a guinea pig FcγRII intracellular segment with an amino acid sequence as shown in SEQ ID NO: 4, and a WW domain with an amino acid sequence as shown in SEQ ID NO: 2; wherein the transmembrane region, the FcγRII intracellular segment, and the WW domain are connected by a linker sequence.

17. A recombinant nucleic acid molecule, characterized in that Encodes the fusion protein according to any one of claims 1 to 16.

18. A recombinant gene expression cassette, characterized in that: Comprising the recombinant nucleic acid molecule of claim 17.

19. The recombinant gene expression cassette according to claim 18, characterized in that: The following elements are included in order from the 5' end to the 3' end: a promoter, a 5'UTR, a nucleic acid encoding a signal peptide, an antigen nucleic acid sequence, a nucleic acid sequence encoding the transmembrane region, a nucleic acid sequence encoding the intracellular segment of FcγRII, a nucleic acid sequence encoding the WW domain, a 3'UTR, and a PolyA tail sequence; wherein the nucleic acid sequence encoding the transmembrane region, the nucleic acid sequence encoding the intracellular segment of FcγRII, and the nucleic acid sequence encoding the WW domain are connected by a linker sequence.

20. A recombinant vector, characterized in that: Comprising the recombinant nucleic acid molecule of claim 17 or the recombinant gene expression cassette of claim 18 or 19.

21. A recombinant host cell, characterized in that Comprising the recombinant nucleic acid molecule of claim 17, or the recombinant gene expression cassette of claim 18 or 19, or the recombinant vector of claim 20.

22. A pharmaceutical composition, characterized in that Comprising one or more fusion proteins according to any one of claims 1 to 16, and / or one or more recombinant nucleic acid molecules according to claim 17, and / or one or more recombinant gene expression cassettes according to claim 18 or 19, and / or one or more recombinant vectors according to claim 20, and / or one or more recombinant host cells according to claim 21.

23. The pharmaceutical composition according to claim 22, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

24. A recombinant vaccine, characterized in that Comprising one or more fusion proteins according to any one of claims 1 to 16, and / or one or more recombinant nucleic acid molecules according to claim 17, and / or one or more recombinant gene expression cassettes according to claim 18 or 19, and / or one or more recombinant vectors according to claim 20, and / or one or more recombinant host cells according to claim 21, and / or one or more pharmaceutical compositions according to claim 22 or 23.

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