Construction and application of fusion protein vaccine platform

Abstract

To provide vaccine platforms that enhance biological response to viral, bacterial, or tumor antigens.SOLUTION: The present invention provides a vaccine comprising a fusion protein containing an interferon-target antigen-immunoglobulin Fc region (or antibody) and a Th cell helper epitope. The present invention also relates to the use of a fusion protein containing an interferon-target antigen-immunoglobulin Fc region (or antibody) and a Th cell helper epitope for preparing a preventive or therapeutic composition. The vaccine of the present invention is produced by a eukaryotic cell expression system, and wild-type and various mutant antigen vaccines can be prepared, and a strong immune response can be induced in the living body by inoculation via an immune route such as subcutaneous / muscle or nasal cavity. The vaccine of the present invention can be used as a preventive or therapeutic vaccine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the fields of genetic engineering and biomedical technology, and specifically to a vaccine, for example, a vaccine comprising a fusion protein containing an interferon-target antigen-immunoglobulin Fc region (antibody) as a backbone. The vaccine of the present invention is used as a vaccine platform to prevent hepatitis B virus (HBV) infection. , na and treatment of chronic hepatitis B (CHB) infection, HB V-kan It is used to treat multiple tumors. [Background technology]

[0002] Approximately 257 million people worldwide are chronically infected with the virus, and approximately 88,700 people die each year from HBV-related end-stage liver disease, including liver failure, cirrhosis, and hepatocellular carcinoma. [1-3] Approximately 30% of cases of liver cirrhosis are caused by HBV, and approximately 40% of cases of hepatocellular carcinoma (HCC) are caused by HBV. Hepatitis B virus infection remains a major public health problem worldwide. [4] However, there is still no effective treatment for chronic hepatitis B. Existing HBV treatments mainly include antiviral drugs (nucleoside / nucleotide analogs) and interferon. While these have some therapeutic effects, they usually fail to induce an effective immune response and therefore cannot completely eliminate HBV infection. Furthermore, long-term medication has relatively significant side effects, and antiviral drugs can also develop drug resistance. Chronic HBV infection is one of the major diseases threatening human health. Therefore, there is an urgent need to explore effective immunotherapy strategies for chronic hepatitis B. The development of a therapeutic vaccine for chronic hepatitis B is of great social and economic importance.

[0003] When an antigen is linked to the Fc region of an immunoglobulin, its half-life is significantly extended and the Fc region of the immunoglobulin can bind to Fc receptors on the surface of antigen-presenting cells, facilitating their processing and presentation by antigen-presenting cells. [5-7]Type I interferons are antiviral cytokines with many biological activities, one of which is the stimulation of immune cells. [8] IFNα can potently induce the differentiation and activation of human DC cells. [9] After type I interferon acts on immature DCs, it promotes the expression of MHC molecules and costimulatory molecules, such as MHC class I, CD80, and CD86, on the DC cell surface, thereby enhancing the ability of DCs to activate T cells. [10-12] It has been reported that type I interferons can enhance the antigen-presenting capacity of DCs after infection with vaccinia virus and lymphocytic chorioencephalitis virus (LCMV). [13-15] Furthermore, type I interferons, after acting on DCs, can promote the migration of DCs to lymph nodes by upregulating the expression of chemokine receptors, thereby promoting T cell activation. [16、17] Recently, more and more studies have shown that type I interferons can be used as immune adjuvants. Le Bon et al. showed that when mice were immunized with a weak immunogen, type I interferons exhibited a strong immune adjuvant effect in mice, inducing long-lasting antibodies and immune memory.

[18] We also found that DC cells are the main cell population on which type I interferon acts. Therefore, antibodies can be used to target and deliver vaccines to DCs, stimulating their activation and cross-presentation function, further enhancing vaccine activity and efficacy.

[0004] The present invention includes Anti There is a need to provide a vaccine platform that enhances the body's response to the gen. Summary of the Invention

[0005] Vaccines are an effective method for preventing and controlling infectious disease outbreaks, and there are various types of vaccines, one of which is protein subunit vaccines. Generally, simple protein subunit vaccines generally have low immunogenicity, which often limits the use of protein subunit vaccines. Therefore, a universal protein subunit vaccine platform is urgently needed. Based on the effects of immunoglobulin Fc region and type I interferon on the immune system, the present inventors have developed a novel vaccine platform for viral infections. Anti Interferon α-viral antiviral drugs are used to enhance the body's response to the virus. original- The present inventors have specifically proposed an immunoglobulin Fc region fusion protein vaccine platform. The present invention provides a type I interferon-protein antigen-immunoglobulin Fc vaccine platform, in which type I interferon acts on antigen-presenting cells to mature and migrate, better fulfilling the roles of antigen presentation and T cell activation. Meanwhile, the Fc portion of the vaccine platform binds to Fc receptors on the surface of antigen-presenting cells, promoting antigen uptake by the antigen-presenting cells and further supporting their function. The present inventors have proposed the fusion of a Th cell helper epitope, which can further improve the immune response effect of type I interferon-protein antigen-immunoglobulin Fc vaccines and is an important component of the vaccine. The present inventors have proposed that the use of antibodies such as anti-PD-L1 to replace the Fc domain can target and deliver the vaccine to DCs, stimulating DC activation and cross-presentation function, thereby further enhancing vaccine activity and efficacy. The present invention provides a novel vaccine platform for the treatment of viral infections. dye It can be used as a preventive and therapeutic vaccine for any disease.

[0006] In some embodiments, the present invention provides a vaccine comprising a fusion protein (Th epitope-bearing) containing an interferon-target antigen-immunoglobulin Fc region (or antibody). In some embodiments, the present invention further provides use of a fusion protein (Th epitope-bearing) containing an interferon-target antigen-immunoglobulin Fc region (or antibody) for preparing a prophylactic or therapeutic composition or kit (e.g., a drug or vaccine composition or kit). The vaccines of the present invention are produced in a eukaryotic cell expression system and can be administered via immunization routes such as subcutaneous / muscle or intranasal. The antibody (abbreviated as Ab) as the structural unit of the fusion polypeptide of the present invention is not particularly limited and may include, for example, a complete antibody, an antibody fragment such as an antibody heavy chain or light chain, or a single-chain antibody, or a DC-targeting, activating antibody, including antibodies such as anti-PD-L1, anti-DEC205, and anti-CD80 / 86.

[0007] In some embodiments, the target antigens described herein are not particularly limited and can be any suitable antigen. In some embodiments, the target antigens described herein can be, for example, ,cormorant Il with the antigen could be.

[0008] In some embodiments, the target antigens used in the vaccines provided herein can be, for example, mutant target antigens that differ from the wild type. In some embodiments, the target antigens described herein can be, for example, mutant target antigens that differ from the wild type. ,cormorant Il of the antigen may be mutant Book As used herein, a wild-type target antigen refers to an immunogenic protein expressed by a virus or other infectious agent or tumor that is encoded by a wild-type gene (a wild-type gene refers to a naturally predominant allele, which is often used as a standard control gene in biological experiments). )of As used herein, a mutant target antigen (mutant) refers to a mutant viral protein expressed by a mutant virus strain that is encoded by a mutant gene that is mutated from the wild-type gene. vinegar.In some embodiments, the mutant target antigen may include, for example, natural point mutations / deletions / gains / truncations, artificial point mutations / deletions / gains / truncations, any combination of natural or artificial mutations, or subtypes generated after mutation, wherein the target antigen is ,cormorant Il Anti In some embodiments, the target antigen used in the vaccines provided herein is a mutant viral antigen. do. As used herein, unless otherwise specified or clearly limited by context, target antigens referred to herein generally include wild-type target antigens and mutant target antigens.

[0009] The present invention relates to interferon (IFN). Tou Hepatitis B virus Pres1 antigen 、B Hepatitis B surface antigen (HBsAg) antigen or peptide Do) and an immunoglobulin Fc region (or antibody) (Th epitope-tagged). The fusion protein may be a homodimeric protein or a heterodimeric protein. When the fusion protein is a dimer, the structural units of the interferon, target antigen, and immunoglobulin Fc region (or antibody Ab) may be present in a first polypeptide chain and / or a second polypeptide chain. The form in which each structural unit exists is not particularly limited; for example, they may exist simultaneously in one chain, or any one or more structural units may exist in one chain and another one or more structural units may exist in another chain.

[0010] The interferon according to the present invention can be selected from the group consisting of type I interferon, type II interferon, and type III interferon, such as IFN-α, IFN-β, IFN-γ, IFN-λ1 (IL-29), IFN-λ2 (IL-28a), IFN-λ (IL-28b), and IFN-ω. The IFN may be of human or murine origin. The interferon according to the present invention is preferably IFN-α (SEQ ID NO: 1, SEQ ID NO: 11 , SEQ ID NO: 12 )

[0011] The immunoglobulin Fc region according to the present invention can be selected from the group consisting of the constant region amino acid sequences of IgG1, IgG2, IgG3, IgG4 and / or IgM, and preferably IgG1 (SEQ ID NO: 2, SEQ ID NO: 13 , SEQ ID NO: 14 )

[0012] The fusion polypeptide of the present invention may also optionally contain one or more Th cell helper epitopes and / or linking fragments (linkers). For example, when the fusion protein is a dimer, the fusion protein may also optionally contain one or more Th cell helper epitopes and / or linking fragments in any one or two chains (i.e., the first polypeptide chain and / or the second polypeptide chain) of the homodimer or heterodimer. As known to those skilled in the art, each structural unit of the fusion protein can be linked by an appropriate linking fragment (linker). The linking fragment that can be used in the vaccine of the present invention is not particularly limited and can be any suitable peptide fragment known in the art. The linking fragment of each structural unit described in the present invention may be a flexible polypeptide sequence, such as linking fragments 1 and 2, for example, SEQ ID NO: 4, SEQ ID NO: 15 It may be represented by an amino acid sequence.

[0013] The N-terminus of each polypeptide sequence consisting of each structural unit described in the present invention contains a corresponding signal peptide capable of promoting protein secretion, such as that shown in the amino acid sequence of SEQ ID NO: 5.

[0014] Preferred antigens according to the present invention are the ad subtype (SEQ ID NO: 6), the ay subtype (SEQ ID NO: 16 Hepatitis B Pres1 antigen, including adr subtype (SEQ ID NO: 7), adw subtype (SEQ ID NO: 17 ), ayw subtype (SEQ ID NO: 18 HBV HBsAg antigen (each subtype and peptide) Do) Includes.

[0015] The homodimeric protein according to the present invention comprises a first polypeptide and a second polypeptide, which are completely identical to each other. The first polypeptide and the second polypeptide are, in order from the N-terminus to the C-terminus, IFN. -cormorant virus antigen (hepatitis B Pres1 antigen or HBsAg anti original) - an immunoglobulin Fc region or a Pan epitope-containing polypeptide, having SEQ ID NO: 8, 19, 22, 25, 26, 28, or 31 The amino acid sequence is represented by the formula:

[0016] The heterodimer according to the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same polypeptide, and the first polypeptide is, in order from C-terminus to N-terminus, an IFN-immunoglobulin Fc region, each having SEQ ID NO: 9, 20, 23, 26, 29, or 32 and the second polypeptide comprises, in order from the C-terminus to the N-terminus, an amino acid sequence represented by Reu virus antigen (hepatitis B Pres1 anti- original) - an immunoglobulin Fc region, having SEQ ID NO: 10, 21, 24, 27, 30, or 33 The amino acid sequence is represented by the formula:

[0017] The present invention also provides the above-mentioned IFN -cormorant Hepatitis B virus antigen (Hepatitis B Pres1 antigen, HBsAg antigen or peptide Do) -Provides the amino acid sequence encoding the immunoglobulin Fc vaccine platform.

[0018] The present invention also relates to nucleotide segments encoding said vaccine platforms and fusion proteins.

[0019] The present invention also relates to a method for preparing the fusion protein or vaccine platform, for example, the method for preparing the fusion protein or vaccine platform, comprising: (1) constructing an expression vector containing a coding gene encoding the fusion protein or vaccine platform, preferably, the expression vector is a pEE12.4 expression vector; (2) constructing a host cell containing the expression vector by transiently transfecting the host cell, preferably the host cell is a 293F cell; (3) culturing the host cells and collecting the cell supernatant; (4) purifying the fusion protein or vaccine platform by Protein A / G affinity chromatography column purification;

[0020] The present invention also includes applications of the vaccine platform, which can be used as a hepatitis B preventive vaccine, and the vaccine platform can be used as a hepatitis B therapeutic vaccine. Use It can be used.

[0021] The present invention includes adjuvants used in the vaccine platform, including aluminum adjuvant (Alum), Toll-like receptor 4 activator ligand MPLA, Toll-like receptor 9 ligand, M59, oligodeoxynucleotide (CpG-ODN), and Freund's adjuvant.

[0022] The present invention includes the clinical use of the combination of said vaccine platform as an HBV therapeutic vaccine with the hepatitis B virus envelope protein HBsAg vaccine in the treatment of chronic hepatitis B virus infection.

[0023] The present invention includes the clinical use of the combination of said vaccine platform with a nucleoside or nucleotide analogue as an HBV therapeutic vaccine in the treatment of chronic hepatitis B virus infection.

[0024] The present invention is a HB V prediction and the use of said vaccine platform in combination with antiviral drugs and other therapies, such as as a preventative or therapeutic vaccine for HBV. V-kan This includes the combined use of said vaccine platform with antiviral and tumor drugs and therapies as a combined tumor preventative or therapeutic vaccine.

[0025] The present invention includes multivalent combination vaccines consisting of the vaccine platform as one component of a vaccine with another virus or pathogen or tumor vaccine.

[0026] The present invention is S This includes adenovirus vaccines or mRNA vaccines or inactivated vaccines or DNA vaccines and any fusion protein vaccines of said vaccine platforms, immunized in a sequential or simultaneous immunization procedure.

[0027] The present invention provides full-length and optionally truncated sequences of said vaccine platform antigens. Column include.

[0028] The present invention relates to mutant sequences such as natural point mutations / deletion mutations / truncations, any combination of natural point mutations, subtypes generated after mutation, and artificial point mutations / deletion mutations / truncations constructed by the present inventors to enhance vaccine efficacy. ,before Any possible variant of the fusion protein vaccine antigen is included.

[0029] The present invention also provides multivalent combination vaccines comprising any of the vaccines of the present invention as one component of a vaccine and another vaccine of the present invention or another vaccine different from the vaccine of the present invention, such as another virus or pathogen or tumor vaccine. N For example, any vaccine of the present invention may contain the same virus. S Adenovirus vaccines or mRNA vaccines or inactivated vaccines or DNA vaccines, and sequential or simultaneous immunization procedures Exemption When used in combination, the combination vaccines can be prepared as convenient kits, as is known in the art.

[0030] Compared with the prior art, the present invention has the following beneficial effects, including but not limited to:

[0031] 1. IFN provided by the present invention -cormorant In the virus antigen-immunoglobulin Fc (or antibody) vaccine platform, the antigen can be varied with multiple components. tree, It may also be a virus-specific antigen, thereby increasing the flexibility and range of use of the vaccine platform.

[0032] 2. IFN provided by the present invention -cormorant In the virus antigen-immunoglobulin Fc (or antibody) vaccine platform, interferon (IFN) promotes the migration and maturation of antigen-presenting cells and increases the expression of costimulatory molecules, thereby favoring antigen presentation to T cells. At the same time, the Fc region (or antibody) of the vaccine platform, on the one hand, increases the molecular weight of the antigen and increases its half-life, and on the other hand, the Fc region (or antibody) binds to Fc receptors on the surface of antigen-presenting cells, promoting the processing and presentation of the antigen by the antigen-presenting cells, thereby favoring the production of an immune response.

[0033] 3. IFN provided by the present invention -cormorantThe virus antigen-immunoglobulin Fc (or antibody) vaccine platform is expressed by a eukaryotic HEK293 cell expression system, and the proteins expressed by HEK293 cells are close to natural protein molecules in terms of molecular structure, physical and chemical properties, protein modifications, and protein biological functions.

[0034] 4. IFN provided by the present invention -cormorant The virus antigen-immunoglobulin Fc (or antibody) vaccine platform has two structures, homodimer or heterodimer, which have better selectivity against different antigens.

[0035] 5. IFN provided by the present invention -cormorant The virus antigen-immunoglobulin Fc vaccine platform fuses Th cell helper epitopes, such as Pan epitopes, utilizes DC-targeting antibodies such as anti-PD-L1, and adds various adjuvants that stimulate the immune response, thereby activating DCs, enhancing DC cross-presentation, and generating strong B cell and T cell immune responses.

[0036] 6. IFN provided by the present invention -cormorant The virus antigen-immunoglobulin Fc (or antibody) vaccine platform has broad applications and can be used not only as a prophylactic vaccine but also as a therapeutic vaccine.

[0037] 7. IFN provided by the present invention -cormorant The virus antigen-immunoglobulin Fc (or antibody) vaccine platform can be used alone or in combination with existing HBsAg commercial vaccines, nucleoside / nucleotide analogues, as therapeutic vaccines.

[0038] 8. The vaccine provided by the present invention can be used as a component of a vaccine to form a multivalent combination vaccine with other viruses or pathogens or tumor vaccines.

[0039] 9. Any fusion protein vaccine in the vaccine platform provided by the present invention may be used with the same virus. S Immunization with an adenovirus vaccine or an mRNA vaccine or an inactivated vaccine or a DNA vaccine can be performed in a sequential or simultaneous immunization procedure.

[0040] 10. The present invention provides full length and any truncated sequences of vaccine platform antigens.

[0041] 11. The present invention provides any possible variants of vaccine platform antigens, including mutant sequences such as naturally occurring point mutations / deletions / gains / truncations, any combination of naturally occurring point mutations, subtypes generated after mutation, and artificial point mutations / deletions / gains / truncations constructed by the present inventors to enhance vaccine efficacy.

[0042] The sequence information related to the present invention is shown below. 1. Unit configuration: SEQ ID NO: 1: Mouse mIFNα4 amino acid sequence (mIFNα) CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKE SEQ ID NO: 11 : Human IFNα2 amino acid sequence (hIFNα) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE SEQ ID NO. 12: Human mutant IFNα2 (Q124R) amino acid sequence (hmIFNα) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE SEQ ID NO: 2: Human IgG1-Fc amino acid sequence EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV SEQ ID NO: 13 : Heterodimeric Fc-hole DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 14 : Heterodimeric Fc-knob DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 3: Th helper epitope Pan HLA DR-binding epitope (PADER) amino acid sequence AKFVAAWTLKAAA SEQ ID NO: 4: Linker 1 amino acid sequence: GGGGSGGGGSGGGGS SEQ ID NO: 15 :Linker 2 amino acid sequence: GSGSGS SEQ ID NO: 5: Signal peptide amino acid sequence: MARLCAFLMILVMMSYYWSACSLG SEQ ID NO: 6: Amino acid sequence of HBV Pres1 (ad subtype) MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVSTIPPPASTNRQSGRQPTTPISPPLRDSHPQA SEQ ID NO: 16 : Amino acid sequence of HBV Pres1 (ay subtype) MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHHPQA SEQ ID NO: 7: Amino acid sequence of HBV HBsAg (adr subtype) MENTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLLPGT STTSTGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYNILSPFLPLLPIFFCLWVYI SEQ ID NO: 17 : Amino acid sequence of HBV HBsAg (adw subtype) MENITSGLLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLSFLGEAPVCLGQNSQSPTRNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGS TTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMIWYWGPSLYSIVCPFTPLLQIFCCLWVFI SEQ ID NO: 18 : Amino acid sequence of HBV HBsAg (ayw subtype) MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQSSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPG SSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVY I 2 Murine-derived IFN vaccine mIFNα-antigen-Fc sequence: SEQ ID NO: 8 : mIFNα-Pres1-Fc amino acid sequence in the homodimer CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKH SLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPA NPPPASTNRQTGRQPTPLSPPLRNTHPQAFEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKH V S EQ ID NO: 9 : First chain mIFNα-Fc-hole amino acid sequence in the heterodimer CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 10 : Amino acid sequence of the second chain Pres1-Fc-knob in the heterodimer mIFNα-Pres1-Fc MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 3 Vaccine IFNα-Pan-Antigen-Fc sequence containing the Pan epitope of murine IFN: SEQ ID NO: 19 : mIFNα-Pan-Pres1-Fc amino acid sequence in the homodimer CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSL CAWEVIRAEVWRALSSSTNLLARLSEEKEGGGGSGGGGSGGGGSRTAKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSP QAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPG K S EQ ID NO: 20 : First chain mIFNα-Fc-hole amino acid sequence in the heterodimer CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 21 : Amino acid sequence of the second chain Pan-Pres1-Fc-knob in the heterodimer mIFN-Pan-Pres1-Fc AKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 4 Human IFN vaccine hIFNα-antigen-Fc sequence: SEQ ID NO: 22 : hIFNα-Pres1-Fc amino acid sequence in the homodimer CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLY LKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQT LPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPG K S EQ ID NO: 23 : First chain hIFN-Fc-hole amino acid sequence in the heterodimer CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSV FLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFKLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 24 : Amino acid sequence of the second chain Pres1-Fc-knob in the heterodimeric hIFNα-Pres1-Fc MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 5 . Vaccine containing the Pan epitope of human IFNα-Pan-Antigen-Fc sequence: SEQ ID NO: 25 : hIFNα-Pan-Pres1-Fc amino acid sequence in the homodimer CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLY LKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQ TLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPG K S EQ ID NO: 26 : First chain hIFNα-Fc-hole amino acid sequence in the heterodimer CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGV GVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSV FLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 27 : Amino acid sequence of the second chain Pan-Pres1-Fc-knob in the heterodimer hIFNα-Pan-Pres1-Fc AKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 6 Human mutant IFN vaccine hmIFNα-Pan-Antigen-Fc sequence: SEQ ID NO: 28: hmIFNα-Pres1-Fc amino acid sequence in the homodimer CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLY LKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQT LPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPG K S EQ ID NO: 29 : Amino acid sequence of the first chain hmIFN-Fc-hole in the heterodimer CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGV GVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSV FLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 30 : Amino acid sequence of the second chain Pres1-Fc-knob in the heterodimer hmIFNα-Pres1-Fc MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 7 Vaccine containing the Pan epitope of human mutant IFNα: hmIFNα-Pan epitope-antigen-Fc sequence SEQ ID NO: 31: hmIFNα-Pan-Pres1-Fc amino acid sequence in the homodimer CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLY LKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQ TLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPG K S EQ ID NO: 32 : First chain hmIFNα4-Fc-hole amino acid sequence in the heterodimer CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGV GVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSV FLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 33 : Amino acid sequence of the second chain Pan-Pres1-Fc-knob in the heterodimer hmIFNα-Pan-Pres1-Fc AKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K [Brief explanation of the drawings]

[0043] [Figure 1]Figure 1. Schematic diagram of the vaccine platform combining the following order in the form of a homodimer: interferon-linked fragment 1-target antigen-immunoglobulin Fc (or antibody); [Figure 2] Figure 2. Schematic diagram of the vaccine platform, combining interferon-linked fragment 1-IgG1-hole, target antigen-IgG1-knob (or antibody) in the form of heterodimers in this order; [Figure 3] Figure 3. Schematic diagram of the vaccine platform, combining interferon-linked fragment 1-IgG1-knob and target antigen-IgG1-hole (or antibody) in the form of heterodimers, respectively; [Figure 4] Figure 4. Schematic diagram of the vaccine platform, combining the following sequence in the form of a homodimer: interferon-linked fragment 1, Th cell helper epitope-linked fragment 2, target antigen, and immunoglobulin Fc (or antibody); [Figure 5] Figure 5. Schematic diagram of vaccine platforms combined in the form of heterodimers in the order of interferon-linked fragment 1-IgG1-hole and Th cell helper epitope-linked fragment 2-target antigen-IgG1-knob (or antibody), respectively; [Figure 6] Figure 6. Schematic diagram of the vaccine platform, combining interferon-linked fragment 1-IgG1-knob and Th cell helper epitope-linked fragment 2-target antigen-IgG1-hole (or antibody) in the form of heterodimers, respectively; [Figure 7] Figure 7. Identification of Pres1-Fc and IFN-Pres1-Fc native proteins by SDS-PAGE electrophoresis. [Figure 8]Figure 8. Compared to free preS1, the fusion proteins preS1-Fc and IFN-preS1-Fc significantly enhance the immunogenicity of antigenic molecules and induce the production of broadly neutralizing antibodies. (a) C57 / BL6 mice (n = 8 / group) were subcutaneously inoculated with free hepatitis B Pres1, Pres1-Fc, or IFNα-Pres1-Fc proteins, and serum Pres1-specific antibody levels were detected by ELISA at the indicated times. (b) Three HBV genotype stable carrier mice (n = 4) were intravenously injected with serum from IFNα-Pres1-Fc protein-immunized mice, and changes in serum Pres1 antigen levels were detected 12 hours later. [Figure 9] Figure 9. IFNα-Pres1-Fc can be used as a preventive vaccine for hepatitis B. C57 / BL6 mice were subcutaneously inoculated with free hepatitis B Pres1, Pres1-Fc, or IFNα-Pres1-Fc proteins and then infected with 1x10 μg of AAV-HBV1.3 virus via tail vein injection 28 days after inoculation. (a) Serum Anti-Pres1 levels before and 1, 2, 3, and 4 weeks after inoculation. (b) Serum Pres1 levels were detected at the indicated time points. (c) Serum HBsAg levels were detected by ELISA at 1, 2, 3, and 4 weeks after inoculation. (d) Percentage of HBsAg-positive mice after AAV-HBV1.3 virus inoculation. [Figure 10] Figure 10. IFNα-Pres1-Fc was used as a preventive vaccine for chronic type B infection. C57 / BL6 mice were infected with 1x10 μg of AAV-HBV1.3 virus via tail vein injection. Six weeks after infection, stably infected mice (n=8 / group) were selected and immunized subcutaneously with recombinant Pres1 and IFNα-Pres1-Fc proteins once every two weeks for a total of three immunizations. (a) Detection of anti-Pres1 antigen in serum; (b) Detection of Pres1 antigen in serum; (c) Detection of the HBV-associated antigen HBsAg levels in mouse serum. [Figure 11]Figure 11. Th cell helper epitopes enhanced antibody responses to the IFNα-Pres1-Fc vaccine. Compared with IFN-preS1-Fc, IFN-Pan-preS1-Fc significantly enhanced the immunogenicity of antigenic molecules. C57 / BL6 mice (n=8 / group) were subcutaneously inoculated with aluminum-free hepatitis B Pres1, Pres1-Fc, or IFNα-Pres1-Fc proteins, and serum Pres1-specific antibody levels were detected by ELISA at the indicated time points. [Figure 12] Figure 12. IFNα-Pan-Pres1-Fc was used as a therapeutic vaccine for chronic type B infection. C57 / BL6 mice were infected with 1x10 μg of AAV-HBV1.3 virus via tail vein injection. Six weeks after infection, stably infected mice (n=8 / group) were selected and subcutaneously inoculated with recombinant Pres1 and IFNα-Pres1-Fc proteins. The mice were immunized once every two weeks for a total of three times. (a) Detection of anti-Pres1 antigen in serum; (b) Detection of Pres1 antigen in serum; (c) Detection of HBV-associated antigen HBsAg levels in mouse serum; (d) Detection of HBV-DNA levels in mouse serum by QPCR. [Figure 13] Figure 13. Combining IFNα-Pres1-Fc with a commercial HBsAg vaccine can break HBsAg-induced immune tolerance and induce HBsAg-HBsAb serological conversion. HBV carrier mice were subcutaneously immunized with IFNα-Pres1-Fc and a commercial HBsAg vaccine, once every two weeks for a total of three immunizations. (a) Pres1 levels in the serum of HBV carrier mice; (b) HBsAg levels; (c) Anti-Pres1 levels in the serum; (d) Anti-HBsAg levels in the serum; (e) HBV-DNA levels in the serum. ***, p<0.001 DETAILED DESCRIPTION OF THE INVENTION

[0044] In order to clarify the objectives, technical solutions and advantages of the present invention, the present invention will be described in detail below with reference to the embodiments and accompanying drawings. The described examples are only illustrative of the present invention and are not intended to limit the scope of the present invention, and the examples are only a part of the present invention, but are not all examples of the present invention. The scope of the present invention is defined by the appended claims.

[0045] Example 1. Vaccine platform design The interferon-target antigen-immunoglobulin Fc (or antibody) structural unit vaccine platform consists of three structural units: the first structural unit is the interferon moiety, the second structural unit is the immunoglobulin Fc region (or antibody), and the third structural unit is the target antigen. In actual construction, the three structural units can be arranged and combined in any configuration, and the target antigen can be linked to a Th cell helper epitope via linker sequence 2. A typical configuration is as follows:

[0046] FIG. 1 is a schematic diagram of the vaccine platform, which combines interferon-linked fragment 1-target antigen-immunoglobulin Fc in the form of a homodimer in this order.

[0047] FIG. 2 is a schematic diagram of the vaccine platform, in which the interferon-linked fragment 1-IgG1-hole and target antigen-IgG1-knob are combined in the form of heterodimers, respectively.

[0048] FIG. 3 is a schematic diagram of the vaccine platform, which combines interferon-linked fragment 1-IgG1-knob and target antigen-IgG1-hole in the form of a heterodimer.

[0049] Next, the target antigen was linked to a cellular helper epitope by linking fragment 2, which was then combined with the other two vaccine platform components, the representative forms of which were as follows:

[0050] FIG. 4 is a schematic diagram of the vaccine platform, in which interferon-linked fragment 1, Th cell helper epitope-linked fragment 2, target antigen, and immunoglobulin Fc are combined in the order in the form of a homodimer.

[0051] FIG. 5 is a schematic diagram of the vaccine platform, in which the interferon-linked fragment 1-IgG1-hole and Th cell helper epitope-linked fragment 2-target antigen-IgG1-knob are combined in the form of heterodimers, respectively.

[0052] FIG. 6 is a schematic diagram of the vaccine platform, in which interferon-linked fragment 1-IgG1-knob and Th cell helper epitope-linked fragment 2-target antigen-IgG1-hole are combined in the order of heterodimers.

[0053] Example 2. Vaccine platform construction, purification, and production Hepatitis B Virus Pres 1Ta We use a protein homodimer as an example to illustrate the expression and production of this vaccine platform.

[0054] 1. Vector construction, host cell transfection, and inducible expression 1.1. Using PEE12.4 as the vector, the vaccine structural unit was constructed into the vector by molecular cloning, and a plasmid capable of expressing the fusion protein was obtained. 293F cells were then transiently transfected, the culture supernatant was collected, and the target protein was finally purified using a protein A affinity chromatography column. Vector construction (e.g., containing HBV preS1) (1) PEE12.4-HindIII-signal peptide 1-interferon-BsiwI-Pres1-BstbI-hIgG1-EcoRI (2 )PEE12.4-HindIII-signal peptide 1-interferon-Bsiwi-PADER-Pres1-hIgG1-EcoR I The linking sequences between each fusion protein fragment were as follows: (1) The linkage fragment 1 was between interferon and Pres1. (2 )stomach The linking sequence between interferon and PADER was linking segment 1, and the linking segment between PADER and Pres1 was linking segment 2.

[0055] 1.2. Rapid expression of target proteins by transient transfection: (1) Cell resuscitation: 3 × 10 Freestyle 293F cells 7 The cells were cryopreserved in CD OptiCHOTM media (containing 10% DMSO) at a concentration of 1000 cells / ml. After removal from liquid nitrogen, they were quickly thawed in a 37°C water bath, added to a 15 ml centrifuge tube containing 10 ml of OptiCHOTM media, and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was suspended in 30 ml of OptiCHOTM media and cultured at 37°C, 8% CO2, and 135 rpm. After 4 days, the cells were expanded, and the concentration of the expanded culture was 3 x 10 6 The cell density was not to exceed 100 cells / ml. (2) Two days before transfection, 0.6–0.8 × 10 6 Suspension culture 293F cells were prepared for transient transfection (200 ml) at a seeding density of 1000 cells / ml. (3) After 2 days, the cell suspension to be transfected was counted to determine the expected cell density of 2.5–3.5 × 10 6 cells / ml, and then the cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. (4) The cells were resuspended in 50 ml of fresh Freestyle 293 media and centrifuged again at 1000 rpm for 5 minutes, and the supernatant was discarded. (5) 293F cells were resuspended in 200 ml of Freestyle 293 media. (6) 600 μg of the plasmid was diluted with 5 ml of Freestyle 293 media, and the bacteria were removed using a 0.22 μM filter. (7) 1.8 mg of PEI was diluted with 5 ml of Freestyle 293 media, and bacteria were removed using a 0.22 μM filter. Immediately after, 5 ml of the plasmid and 5 ml of PEI were mixed and left to stand at room temperature for 5 minutes. (8) The plasmid / PEI mixture was added to the cell suspension, and the mixture was placed in an incubator at 37°C, 8% CO2, and 85 rpm. 50 μg / L of LONG growth factor was added. TM The culture was carried out while supplementing with R3IGF-1. (9) After 4 hours, 200 ml of EX-CELL™ 293 media and 2 mM Glutamine were added, and the rotation speed was set to 135 rpm for continued cultivation. (10) After 24 hours, the cell growth inhibitor 3.8 mM VPA was added, and after 72 hours, 40 ml of medium D was added. After continued cultivation for 6 to 8 days (cell viability less than 70%), the supernatant was collected and stored until it was used in the next purification step.

[0056] 1.3. Recovery and purification of fusion protein and electrophoretic verification

[0057] 2. Purification of target protein using Protein A: (1) Sample preparation: The suspension cell culture was transferred to a 500 ml centrifuge bucket and centrifuged at 8000 rpm for 20 minutes. The precipitate was discarded, and the supernatant was filtered through a 0.45 μM filter to remove impurities. NaN3 was then added to a final concentration of 0.05% to prevent bacterial contamination during purification. Chromatography column assembly: An appropriate amount of Protein A Agarose (calculated by purifying 20 mg of human Fc fusion protein per ml of Protein A) was taken, mixed thoroughly, and added to the chromatography column. The column was left to stand at room temperature for approximately 10 minutes to allow Protein A and the 20% ethanol solution to overlap. The outlet at the bottom was then opened, and the ethanol solution was allowed to slowly drain by gravity. (3) The chromatography column was washed with 10 column volumes of distilled water and binding buffer (20 mM sodium phosphate + 0.15 M NaCl, pH 7.0) and equilibrated. (4) Using a constant flow pump, the sample was loaded at a flow rate of 10 column volumes / hour, the flow-through was collected, and the sample loading was repeated twice. (5) The column was washed with 10 or more column volumes of binding buffer to remove impurities until no protein was detected in the effluent. (6) Elution was performed using Elution Buffer (0.1 M Glycine, pH 2.7), and each 1 ml of the solution was collected in a separate tube. The elution peaks were observed using a protein indicator solution (Bio-Rad protein assay). The collected tubes containing the elution peaks were mixed and neutralized with an appropriate amount of 1 M Tris, pH 9.0 (pH was adjusted to 6-8, 0.5 or more higher than the isoelectric point of the purified protein). (7) Using a Zeba desalting and concentration centrifugal column, the target protein solution was replaced with the target buffer solution (the pH of the buffer solution was adjusted to avoid the isoelectric point of the protein). Protein concentration was determined by SDS-PAGE electrophoresis and NanoDrop2000 using BSA as a standard. (8) After elution, the column was washed sequentially with 20 column volumes of distilled water and 10 column volumes of 20% ethanol. Finally, the ethanol solution was immersed in a gel medium and stored at 4°C.

[0058] 3. Identification of the protein by SDS-PAGE electrophoresis is shown in FIG.

[0059] Example 3. Compared with simple Pres1 antigen, FNα-Pres1-Fc, Pres1-Fc can induce stronger immune responses in mice.

[0060] Materials: C57BL / 6 male mice (5-8 weeks old) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was purchased from Beijing Kangwei Biological Technology Co., Ltd., a 96-well ELISA measurement board was purchased from Corning Costar, ELISA colorimetric solution was purchased from eBioscience, and the microplate reader used was SPECTRA max PLUS 384 from Molecular, USA. The aluminum adjuvant used was purchased from Sigma.

[0061] method: (1) Immunization of mice with Pres1 fusion proteins: Mice were subcutaneously immunized with 80 pmol of IFN-Pres1-Fc or 80 pmol of Pres1-Fc or Pres1 protein mixed with aluminum adjuvant. Antibody detection was performed by collecting mouse sera by retro-orbital bleeding at the indicated time points. (2) Antibodies produced by IFNα-Pres1-Fc have broad neutralizing effects against HBV of different genotypes. Five-week-old male C57BL / 6 mice were injected with 1x10 AAV-HBV 1.3 (HBV genotypes B, C, and D) via the tail vein. 11 Mice were infected with 1 μg of virus, and six weeks later, mice with stable HBV antigen expression were screened and tested. The screened mice (4 mice per group) were intravenously injected with 200 μl of serum from IFNα-Pres1-Fc-immunized mice. After 12 hours, serum samples were collected, and changes in Pres1 antigen expression in the mice before and after antiserum administration were analyzed by ELISA. (3) Detection of anti-Pres1 specific antibodies in serum by ELISA. 50 μl of Pres1 (2 μg / ml) coating solution was added to an ELISA plate (Corning 9018) at 4°C overnight. The plate was washed once with 260 μl of PBS per well and blocked with 5% blocking solution (5% FBS) at 37°C for 2 hours. Serum samples (1:10, 1:100, 1:1000, 1:10000) were diluted with PBS and added to the blocked ELISA plate at 50 μl / well and incubated at 37°C for 1 hour. After washing five times with 260 μl of PBST, 50 μl of enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted in PBS) was added to each well and incubated at 37°C for 1 hour. Wash five times with 260 μl of PBST each time, add 100 μl of the substrate TMB per well, incubate at room temperature away from light, wait for the color development of the substrate, add 50 μl of termination solution (2N H2SO4) to each well to terminate the color development, and read the plate with a microplate reader to find that the OD was 450-630.

[0062] Results: Free Pres1 was weakly immunogenic. However, the addition of IFNα and Fc moieties to Pres1 to form the IFNα-Pres1-Fc fusion protein significantly improved its immunogenicity, as shown in Figure 8(a). Furthermore, as shown in Figure 8(b), the antibodies produced by IFNα-Pres1-Fc had broad neutralizing effects against different HBV genotypes.

[0063] Example 4. IFNα-Pres1-Fc can be used as a prophylactic vaccine against hepatitis B. Materials: C57BL / 6 (6-8) week old male mice were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd., and HBsAg detection kits were purchased from Shanghai Kehua Biotechnology Co., Ltd. AAV-HBV 1.3 virus was purchased from Guangzhou Paijin Biotechnology Co., Ltd. Other experimental materials were the same as in Example 3.

[0064] method: (1) Mice were subcutaneously immunized with 80 pmol of different forms of Pres1 vaccine, including simple Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins. Serum was collected 28 days after immunization and analyzed in 1x10 11 Mice were infected with 1 μg AAV-HBV 1.3 virus, and serum samples were collected weekly for 4 consecutive weeks. Anti-Pres1 antibody, HBsAg, and Pres1 antigen levels were measured in the serum. At week 3, peripheral HBV DNA levels were measured. (2) Detection of Pres1-specific antigen in serum by ELISA. Antigen coating: Pres1 antibody XY007 (4 μg / ml) coating solution was added to an ELISA plate (Corning 9018) at 50 μl / well and coated overnight at 4°C. The plate was washed once with 260 μl of PBS per well and blocked with 5% blocking solution (5% FBS) at 37°C for 2 hours. Serum samples (1:10, 1:100) were diluted with PBS and added to the blocked ELISA plate at 50 μl / well, with two replicate wells per dilution, and incubated at 37°C for 1 hour. After washing five times with 260 μl of PBST, 50 μl of enzyme conjugate (from the Corning HBsAg Detection Kit) was added to each well and incubated at 37°C for 1 hour. Wash five times with 260ul of PBST each time, add 100ul of substrate TMB per well, incubate at room temperature away from light, wait for the color development of the substrate, add 50ul of termination solution (2N H2SO4) to each well to stop the color development, and read the plate with enzyme marker, OD450-630.

[0065] Results: As shown in Figure 9(a), mice in the IFNα-Pres1-Fc immunization group produced high levels of Pres1 antibodies before virus challenge and maintained these levels throughout the virus infection. Immunization with the IFN-Pres1-Fc vaccine significantly prevented HBV infection compared with the non-immunized group. The anti-preS1 antibodies produced after immunization rapidly and completely eliminated preS1 antigen in serum (Figure 9(b)). Furthermore, most of the IFN-Pres1-Fc immunization mice infected with the virus showed negative peripheral HBsAg (Figures 9(c) and 9(d)). The above experimental results, as shown in Figure 9, demonstrate that IFN-Pres1-Fc as a vaccine can effectively prevent HBV infection.

[0066] Example 5. IFNα-Pres1-Fc was used as a therapeutic vaccine for chronic type B infection. Materials: C57BL / 6 male mice (4 weeks old) were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd. AAV-HBV 1.3 was purchased from Guangzhou Paijin Biotechnology Co., Ltd. HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd. Other experimental materials were the same as in Example 4.

[0067] method: (1) Screening of HBV carrier mice: 4-week-old HBV C57BL / 6 mice were injected with 1x10 11 Mice were injected with μg AAV-HBV 1.3 virus, and the HBV antigen HBsAg was detected 1 to 6 weeks later. Mice with stable HBsAg expression were screened and used as HBV carrier mice. (2) Screened mice were subcutaneously injected with 80 pmol of different forms of Pres1 protein once every two weeks for a total of three immunizations. Mouse serum was collected 14 days after immunization and then weekly. The levels of anti-Pres1 antibody, HBsAg, and Pres1 antigen in the mouse serum were detected by ELISA. After the final blood collection, the HBV-DNA content in the mouse peripheral blood was measured.

[0068] Results: We detected changes in serum preS1 antigen, serum Pres1 antibody, and HBsAg in carrier mice immunized with the IFN-Pres1-Fc vaccine. The results showed that after immunization with the IFNα-Pres1-Fc vaccine, mice produced high levels of anti-Pres1 antibody (Figure 10(a)), and serum preS1 antigen was completely eliminated (Figure 10(b)). At the same time, serum HBsAg also decreased to a certain extent (Figure 10(c)), but neither the untreated control group nor the simple Pres1 vaccine group had a therapeutic effect (Figure 10).

[0069] Example 6. T cell helper epitopes enhanced antibody responses to IFNα-Pres1-Fc vaccine. The materials were the same as in Example 3.

[0070] method: (1) Immunization of mice with Pres1 fusion proteins: Mice were subcutaneously immunized with 80 pmol of IFN-Pan-Pres1-Fc or 80 pmol of IFN-Pan-Pres1-Fc, Pres1-Fc, or Pres1 proteins containing the Pan epitope. Antibody detection was performed by collecting mouse sera by retroorbital bleeding at the indicated time points. (2) Detection of anti-Pres1 specific antibodies in serum by ELISA, same as in Example 3.

[0071] Results: Compared with fusion protein vaccines such as IFN-preS1-Fc, IFN-Pan-preS1-Fc significantly enhanced the immunogenicity of antigen molecules and induced the production of broadly neutralizing antibodies. C57 / BL6 mice (n=8 / group) were subcutaneously inoculated with aluminum-free hepatitis B Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins, and serum Pres1-specific antibody levels were detected by ELISA at the designated times.

[0072] Example 7. IFNα-Pan-Pres1-Fc was used as a preventive vaccine against chronic type B infection. Materials: C57BL / 6 male mice (4 weeks old) were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd. AAV-HBV 1.3 was purchased from Guangzhou Paijin Biotechnology Co., Ltd. HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd. Other experimental materials were the same as in Example 4.

[0073] method: (1) Screening of HBV carrier mice: 4-week-old HBV C57BL / 6 mice were injected with 1x10 11 Mice were injected with μg AAV-HBV 1.3 virus, and the HBV antigen HBsAg was detected 1 to 6 weeks later. Mice with stable HBsAg expression were screened and used as HBV carrier mice. (2) Screened mice were subcutaneously injected with 80 pmol of different forms of Pres1 protein once every two weeks for a total of three immunizations. Mouse serum was collected 14 days after immunization and then weekly. The levels of anti-Pres1 antibody, HBsAg, and Pres1 antigen in the mouse serum were detected by ELISA. After the final blood collection, the HBV-DNA content in the mouse peripheral blood was measured.

[0074] Results: We detected changes in serum preS1 antigen, serum Pres1 antibody, and HBsAg in carrier mice immunized with the IFN-Pan-Pres1-Fc vaccine. The results showed that after IFN-Pan-Pres1-Fc vaccination, mice produced high levels of anti-Pres1 antibody (Figure 12(a)). Furthermore, serum preS1 antigen was completely eliminated (Figure 12(b)), and serum HBsAg was reduced to a certain extent (Figure 12(c)). However, neither the untreated control group nor the simple Pres1 vaccine-immunized group had any therapeutic effect. Furthermore, HBV DNA was significantly reduced in the IFNα-Pan-Pres1-Fc-immunized group (Figure 12(d)).

[0075] Example 8. Combining IFNα-Pan-Pres1-Fc with a commercial HBsAg vaccine can break the immune tolerance caused by HBsAg and induce HBsAg-HBsAb serological conversion. Materials: C57BL / 6 male mice (4 weeks old) were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd. AAV-HBV 1.3 was purchased from Guangzhou Paijin Biotechnology Co., Ltd. HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd. Anti-HBsAg kit was purchased from Beijing Wantai Biopharmaceutical Co., Ltd. Commercially available HBsAg vaccine was purchased from Emei Hanxin Vaccine (Dalian) Co., Ltd. Other experimental materials were the same as in Example 7.

[0076] method: (1) Screening of HBV carrier mice: 4-week-old HBV C57BL / 6 mice were injected with 1x10 11 Mice were injected with μg AAV-HBV 1.3 virus, and the HBV antigen HBsAg was detected 1 to 6 weeks later. Mice with stable HBsAg expression were screened and used as HBV carrier mice. (2) Screened HBV carrier mice were immunized with 80 pmol of IFNα-pan-Pres1-Fc and simultaneously 2 μg of a commercially available HBsAg vaccine twice, 14 days apart. Serum was collected 14 days after the first immunization and weekly thereafter to detect changes in anti-Pres1, Pres1, anti-HBsAg, and HBsAg levels. Finally, HBV-DNA levels were measured at the final serum collection.

[0077] Results: Combining IFNα-Pan-Pres1-Fc with commercially available HBsAg as a therapeutic strategy for chronic hepatitis B can ultimately break the immune tolerance induced by HBsAg. The immune response generated in HBV-resistant mice can completely eliminate preS1 antigen in serum (as shown in Figure 13(a)), and high concentrations of Pres1 antibodies are present in serum (as shown in Figure 13(c)). What is exciting is that the IFN-Pan-Pres1-Fc vaccine can effectively eliminate HBsAg in serum while inducing partial conversion of serological HBsAb (as shown in Figures 13(b) and 14(d)). This is considered to be a clinically important indicator of HBV cure. Furthermore, the expression levels of HBV-associated DNA in peripheral blood were detected using fluorescent quantitative PCR (real-time PCR). Compared to the control group, the immunization regimen combining IFNα-Pan-Pres1-Fc and commercially available HBsAg ultimately reduced peripheral HBV DNA levels (as shown in Figure 13(e)). Based on these results, we devised a vaccine strategy for the treatment of chronic hepatitis B using a vaccine combining IFNα-Pan-Pres1-Fc and commercially available HBsAg.

[0078] References: [1] WHO W. Global hepatitis report 2017 [J]. Geneva: World Health Organization, 2017, [2] RAZAVI-SHEARER D, GAMKRELIDZE I, NGUYEN MH, et al. Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modeling study [J]. The lancet Gastroenterology & hepatology, 2018, 3(6): 383-403. [3] SCHWEITZER A, HORN J, MIKOLAJCZYK R T, et al. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013 [J]. The Lancet, 2015, 386(10003): 1546-55. [4] VOS T, ABAJOBIR A A, ABATE K H, et al. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016 [J]. The Lancet, 2017, 390(10100): 1211-59 . [5 ] KONTERMANN R E. Strategies for extended serum half-life of protein therapeutics [J]. Current opinion in biotechnology, 2011, 22(6): 868-76. [ 6 ] MEKHAIEL D N, CZAJKOWSKY D M, ANDERSEN J T, et al. Polymeric human Fc-fusion proteins with modified effector functions [J]. Scientific reports, 2011, 1(1): 1-11. [ 7] ROOPENIAN D C, AKILESH S. FcRn: the neonatal Fc receptor comes of age [J]. Nature reviews immunology, 2007, 7(9): 715-25. [ 8 ] VILCEK J. Fifty years of interferon research: aiming at a moving target [J]. Immunity, 2006, 25(3): 343-8. [ 9 ] BRACCI L, LA SORSA V, BELARDELLI F, et al. Type I interferons as vaccine adjuvants against infectious diseases and cancer [J]. Expert review of vaccines, 2008, 7(3): 373-81. [ 10 ] HAHM B, TRIFILO M J, ZUNIGA E I, et al. Viruses evade the immune system through type I interferon-mediated STAT2-dependent, but STAT1-independent, signaling [J]. Immunity, 2005, 22(2): 247-57. [ 11 ] ITO T, AMAKAWA R, INABA M, et al. Differential regulation of human blood dendritic cell subsets by IFNs [J]. The Journal of Immunology, 2001, 166(5): 2961-9. [ 12] MONTOYA M, SCHIAVONI G, MATTEI F, et al. Type I interferons produced by dendritic cells promote their phenotypic and functional activation [J]. Blood, 2002, 99(9): 3263-71. [ 13 ] LE BON A, ETCHART N, ROSSMANN C, et al. Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon [J]. Nature immunology, 2003, 4(10): 1009-15. [ 14 ] LE BON A, DURAND V, KAMPHUIS E, et al. Direct stimulation of T cells by type I IFN enhances the CD8+ T cell response during cross-priming [J]. The Journal of Immunology, 2006, 176(8): 4682-9. [ 15 ] SPADARO F, LAPENTA C, DONATI S, et al. IFN-α enhances cross-presentation in human dendritic cells by modulating antigen survival, endocytic routing, and processing [J]. Blood, The Journal of the American Society of Hematology, 2012, 119(6): 1407-17. [ 16] PARLATO S, SANTINI S M, LAPENTA C, et al. Expression of CCR-7, MIP-3β, and Th-1 chemokines in type I IFN-induced monocyte-derived dendritic cells: importance for the rapid acquisition of potent migratory and functional activities [J]. Blood, The Journal of the American Society of Hematology, 2001, 98(10): 3022-9. [ 17 ] ROUZAUT A, GARASA S, TEIJEIRA A, et al. Dendritic cells adhere to and transmigrate across lymphatic endothelium in response to IFN‐α [J]. European journal of immunology, 2010, 40(11): 3054-63. [ 18 ] LE BON A, SCHIAVONI G, D'AGOSTINO G, et al. Type I interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo [J]. Immunity, 2001, 14(4): 461-70.

Claims

1. A vaccine comprising a fusion protein containing an interferon, a target antigen, and an immunoglobulin Fc region as a first structural unit, a second structural unit, and a third structural unit, respectively; The interferon is a first structural unit and has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 11, or SEQ ID NO: 12; the immunoglobulin Fc region is a second structural unit, the target antigen is a third structural unit and is an HBV Pres1 antigen; The fusion protein comprises one or more Th cell helper epitopes and multiple linked fragments.

2. The vaccine described in claim 1, wherein the fusion protein is a homodimer comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain and the second polypeptide chain being identical and each comprising, in order from N-terminus to C-terminus, the interferon, the one or more Th cell helper epitopes, the target antigen and the immunoglobulin Fc region.

3. The vaccine of claim 1, wherein the immunoglobulin Fc region is selected from the Fc regions of IgG1, IgG2, IgG3, IgG4 and IgM, and is preferably an IgG1 Fc region.

4. A vaccine described in any one of claims 1 to 3, wherein the target antigen is an HBV Pres1 antigen represented by the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO:

18.

5. A vaccine described in any one of claims 1 to 4, wherein the Th cell helper epitope is represented by the amino acid sequence of SEQ ID NO:

3.

6. A vaccine described in any one of claims 1 to 5, wherein the fusion protein comprises a linking fragment between each structural unit, and the linking fragment is a flexible polypeptide sequence selected from the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO:

15.

7. A nucleic acid molecule encoding a fusion protein in the vaccine described in any one of claims 1 to 6.

8. An expression vector comprising the nucleic acid molecule of claim 7.

9. A host cell, such as a eukaryotic cell, containing the nucleic acid molecule of claim 7 or the expression vector of claim 8.

10. A vaccine described in any one of claims 1 to 6 for the prevention or treatment of HBV infection or hepatitis B.

11. A composition or kit comprising the vaccine described in any one of claims 1 to 6.

12. A composition or kit described in claim 11 for the prevention or treatment of HBV infection or hepatitis B.

13. A vaccine described in any one of claims 1 to 6 or 10, or a composition or kit described in any one of claims 11 to 12, wherein the vaccine, composition or kit can be administered by an immunization route such as intramuscular, intravenous, transdermal, subcutaneous or nasal, and the vaccine, composition or kit may further comprise an adjuvant, which may include aluminum adjuvant (Alum), Toll-like receptor 4 activator ligand MPLA, Toll-like receptor 9 ligand, oligodeoxynucleotide (CpG-ODN), M59, and Freund's adjuvant.

14. The vaccine of any one of claims 1 to 6 or 10, or the composition or kit of any one of claims 11 to 13, wherein the vaccine can be used in combination with another preventive or therapeutic therapy, for example, the vaccine can be a hepatitis B therapeutic vaccine, and the hepatitis B therapeutic vaccine can be used in combination with another preventive or therapeutic hepatitis B therapy, for example, the hepatitis B therapeutic vaccine can be used in combination with a hepatitis B virus envelope protein HBsAg vaccine, for example, for the treatment of chronic hepatitis B virus infection, for example, the hepatitis B therapeutic vaccine can be used in combination with a nucleoside or nucleotide analogue, for example, for the treatment of chronic hepatitis B virus infection, for example, the vaccine can be combined with other viruses or pathogens or tumor vaccines to form a multivalent combination vaccine, for example, the vaccine is administered in a sequential or simultaneous immunization procedure with an adenovirus vaccine, an mRNA vaccine, an inactivated vaccine, or a DNA vaccine against the same virus.

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