Construction and application of fusion protein vaccine platform

Abstract

The invention relates to construction and application of a fusion protein vaccine platform. The invention provides a vaccine. The vaccine comprises fusion protein of an interferon-target antigen-immunoglobulin Fc region (or antibody) and a Th cell helper epitope. The invention also relates to the use of a fusion protein comprising an interferon-target antigen-immunoglobulin Fc (or antibody) region and a Th cell helper epitope for the preparation of a prophylactic or therapeutic composition. The vaccine provided by the invention can be produced and prepared into wild vaccines and vaccines of various mutant antigens through an eukaryotic cell expression system, and can induce strong immune response of a body through inoculation in immune ways such as subcutaneous / muscle or nasal cavity. The vaccine of the present invention can be used as a prophylactic or therapeutic vaccine.

Description

[0001] This application is a divisional application of the invention patent application with application date of December 31, 2021, application number 202111680865.3, and invention name “Construction and Application of Fusion Protein Vaccine Platform”. Technical Field

[0002] The present invention belongs to the field of genetic engineering and biomedical technology, and specifically relates to a vaccine, such as a fusion protein vaccine containing interferon-target antigen-immunoglobulin Fc region (antibody) as the main skeleton. The vaccine of the present invention can be used as a vaccine platform for preventing hepatitis B virus (HBV) infection and treating chronic hepatitis B (CHB) infection and HBV-related tumors. Background Art

[0003] There are approximately 257 million people with chronic hepatitis B virus infection worldwide, and approximately 88,700 people die each year from end-stage liver disease caused by HBV, including liver failure, cirrhosis, and hepatocellular carcinoma. [1-3] About 30% of liver cirrhosis patients are caused by HBV, and about 40% of hepatocellular carcinoma (HCC) are caused by HBV. [4] . Hepatitis B virus infection remains a major global public health issue. However, there is still no effective strategy for the treatment of chronic hepatitis B. Existing HBV treatments mainly include antiviral drugs (nucleoside / nucleotide analogs) and interferon. Although they have certain therapeutic effects, they usually cannot induce effective immune responses and thus cannot completely eliminate HBV infection. Moreover, long-term medication causes significant side effects, and antiviral drugs can also develop drug resistance. Chronic HBV infection is one of the major diseases that threaten human health. It is urgent to explore effective immunotherapy strategies for chronic hepatitis B. The development of a therapeutic vaccine for chronic hepatitis B has very important social and economic significance.

[0004] When the antigen is linked to the Fc region of an immunoglobulin, the half-life of the antigen will be significantly prolonged, and the Fc region of the immunoglobulin can bind to the Fc receptor on the surface of antigen-presenting cells, thereby promoting the processing and presentation of the antigen by the antigen-presenting cells. [5-7] As an antiviral cytokine, type I interferon has many biological activities, one of which includes the stimulation of immune cells. [8] IFNα can strongly induce the differentiation and activation of human DC cells [9] After acting on immature DCs, type I interferons can promote the expression of MHC molecules and co-stimulatory molecules on the surface of DCs, such as MHC class I, CD80 and CD86, thereby enhancing the ability of DCs to activate T cells. [10-12]。It has been reported that type I interferon can promote the antigen - presenting ability of DCs after infection with vaccinia virus and lymphocytic choriomeningitis virus (LCMV). [13-15] 。In addition, after type I interferon acts on DCs, it can promote the migration of DCs to lymph nodes by up - regulating the expression of chemokine receptors, thereby promoting the activation of T cells. [16, 17] 。Recently, more and more studies have shown that type I interferon can be used as an immune adjuvant. The research by Le Bon et al. showed that when immunizing mice with a weak immunogen, type I interferon exhibited a strong immune adjuvant effect in mice, inducing the production of long - acting antibodies and immune memory.

[18] 。The author also found that the main cell population for type I interferon to exert its effect is DC cells. At the same time, using antibodies to target - deliver vaccines to DCs and stimulate the activation and cross - presentation function of DCs will further enhance the activity and potency of the vaccine.

[0005] There is a need for a vaccine platform in the present invention to enhance the body's response to viral antigens. Summary of the Invention

[0006] Vaccines are an effective way to prevent and control major emerging infectious diseases. There are various types of vaccines, and an important category is protein subunit vaccines. Generally speaking, simple protein subunit vaccines generally have poor immunogenicity, which often limits the use of protein subunit vaccines. Therefore, a general - purpose protein subunit vaccine platform is urgently needed. Based on the effects of the Fc region of immunoglobulin and type I interferon on the immune system, the inventors specifically proposed an interferon - α - viral antigen - immunoglobulin Fc region fusion protein vaccine platform to enhance the body's response to viral antigens. The present invention provides a type I interferon - protein antigen - immunoglobulin Fc vaccine platform. In this platform, type I interferon can act on antigen - presenting cells to make them mature and migrate, so as to better play the role of antigen presentation and activation of T cells. On the other hand, the Fc part of this vaccine platform can bind to the Fc receptor on the surface of antigen - presenting cells, thereby enhancing the uptake of antigens by antigen - presenting cells and further helping antigen - presenting cells play their roles. The inventors proposed that fusing Th cell - helper epitopes can further enhance the immune response effect of the type I interferon - protein antigen - immunoglobulin Fc vaccine, which is an important component of this vaccine. The inventors proposed that antibodies such as anti - PD - L1 can be used to replace Fc to target - deliver the vaccine to DCs and stimulate the activation and cross - presentation function of DCs, which will further enhance the activity and potency of the vaccine. As a new vaccine platform, the present invention can be used as a preventive and therapeutic vaccine for diseases such as viral infections.

[0007] In some embodiments, the present invention provides a vaccine comprising a fusion protein (with Th epitope attached) of interferon-target antigen-immunoglobulin Fc region (or antibody). In some embodiments, the present invention also provides the use of a fusion protein (with Th epitope attached) containing interferon-target antigen-immunoglobulin Fc region (or antibody) for the preparation of a preventive or therapeutic composition or kit (e.g., a drug or vaccine composition or kit). The vaccine of the present invention can be produced by a eukaryotic cell expression system and inoculated through immunization routes such as subcutaneous / muscular or nasal. For the fusion polypeptide of the present invention, the antibody (abbreviated as Ab) as a structural unit is not particularly limited, and may include, for example, a complete antibody or an antibody fragment, such as an antibody heavy chain and a light chain, or a single-chain antibody, and may be a DC-targeted activation antibody, including anti-PD-L1, anti-DEC205, anti-CD80 / 86 and other antibodies.

[0008] In some embodiments, the target antigen described herein is not particularly limited and can be any appropriate antigen. In some embodiments, the target antigen described herein can be, for example, a viral antigen.

[0009] In some embodiments, the target antigen used in the vaccine provided by the present invention may be, for example, a mutated target antigen different from the wild type. In some embodiments, the target antigen described herein may be, for example, a mutant of a viral antigen. In this article, a wild-type target antigen refers to an immunogenic protein expressed by a virus or other infectious agent or tumor encoded by a wild-type gene (a wild-type gene refers to the majority allele in nature, often used as a standard control gene in biological experiments). In this article, a mutated target antigen (mutant) refers to a mutated viral protein expressed by a mutant virus strain encoded by a mutant gene mutated from a wild-type gene. In some embodiments, the mutated target antigen may include, for example, a natural point mutation / deletion mutation / increase mutation / truncation, an artificial point mutation / deletion mutation / increase mutation / truncation, any combination of natural or artificial mutations, and a subtype produced after mutation, wherein the target antigen may be a viral antigen. In some embodiments, the target antigen used in the vaccine provided by the present invention is a mutated viral antigen. In this article, unless otherwise explicitly stated or the context clearly limits, the target antigen mentioned herein generally includes a wild-type target antigen and a mutant target antigen.

[0010] The purpose of the present invention is to provide a vaccine platform, which is composed of interferon (IFN) and viral antigen (hepatitis B virus Pres1 antigen)-immunoglobulin Fc region (or antibody) (additional Th epitope). The fusion protein can be a homologous or heterologous dimeric protein. When the fusion protein is a dimer, the interferon, target antigen, immunoglobulin Fc region (or antibody Ab) as structural units can exist in the first polypeptide chain and / or the second polypeptide chain, and the existence of each structural unit is not particularly limited, for example, it can exist in one chain at the same time, or any one or more structural units can exist in one chain, and another one or more structural units can exist in another chain.

[0011] The interferon of the present invention can be selected from 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 can be from human or mouse sources; preferably type I interferon IFN-α (SEQ ID NO.1, SEQ ID NO.11, SEQ ID NO.12).

[0012] The immunoglobulin Fc region of the present invention can be selected from the constant region amino acid sequence of IgG1, IgG2, IgG3 and IgG4 / or IgM, preferably IgG1 (SEQ ID NO.2, SEQ ID NO.13, SEQ ID NO.14).

[0013] The fusion polypeptide of the present invention may also optionally contain one or more Th cell helper epitopes and / or connecting fragments (linkers). For example, when the fusion protein is a dimer, the fusion protein may optionally contain one or more Th cell helper epitopes and / or connecting 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, the various structural units of the fusion protein may be connected by appropriate connecting fragments (linkers). The connecting fragments that can be used in the vaccine of the present invention are not particularly limited and may be any appropriate peptide fragment known in the art. The connecting fragments of the various structural units of the present invention may be flexible polypeptide sequences, and may be connecting fragments 1 and 2, such as shown in the amino acid sequences of SEQ ID NO.4 and SEQ ID NO.15.

[0014] The N-terminus of the polypeptide sequences composed of the structural units described in the present invention all contain corresponding signal peptides capable of promoting protein secretion, such as shown in the amino acid sequence of SEQ ID NO.5.

[0015] The preferred antigens described in the present invention include hepatitis B Pres1 antigen, including ad subtype (SEQ ID NO.6) and ay subtype (SEQ ID NO.16), HBV HBsAg antigen (various subtypes and peptides), including adr subtype (SEQ ID NO.7), adw subtype (SEQ ID NO.17), and ayw subtype (SEQ ID NO.18).

[0016] The homodimeric protein described in the present invention comprises a first polypeptide and a second polypeptide, and the first polypeptide is identical to the second polypeptide. The sequence of the first polypeptide and the second polypeptide from segment N to segment C is IFN-viral antigen (hepatitis B Pres1 antigen)-immunoglobulin Fc region; or a polypeptide containing Pan epitope. It comprises the amino acid sequence shown in SEQ ID NO.8, 19, 22, 25, 26, 28, 31.

[0017] The heterodimer of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same polypeptide, the first polypeptide is an IFN-immunoglobulin Fc region from the C segment to the N terminus, comprising the amino acid sequences shown in SEQ ID NOs. 9, 20, 23, 26, 29, and 32; the second polypeptide is a viral antigen (hepatitis B Pres1 antigen)-immunoglobulin Fc region from the C segment to the N segment; comprising the amino acid sequences shown in SEQ ID NOs. 10, 21, 24, 27, 30, and 33.

[0018] The present invention also provides an amino acid sequence encoding the above IFN-hepatitis B Pres1 antigen, HBsAg antigen or peptide-immunoglobulin Fc vaccine platform.

[0019] The present invention also relates to nucleotide fragments encoding the vaccine platform and fusion protein.

[0020] The present invention also relates to a method for preparing the fusion protein or vaccine platform, for example, the preparation method comprises the following steps:

[0021] (1) constructing an expression vector comprising the gene encoding the fusion protein or vaccine platform, preferably, the expression vector is a pEE12.4 expression vector;

[0022] (2) constructing a host cell containing the expression vector by transiently transfecting the host cell. Preferably, the host cell is a 293F cell;

[0023] (3) culturing the host cells and collecting the cell supernatant;

[0024] (4) Purifying the fusion protein or vaccine platform by purifying the protein using a Protein A / G affinity chromatography column.

[0025] The present invention further includes the use of the vaccine platform, which can be used as a hepatitis B preventive vaccine and the use of the vaccine platform as a hepatitis B therapeutic vaccine.

[0026] The present invention comprises the adjuvant used by the vaccine platform, wherein the adjuvant comprises aluminum adjuvant (Alum), Toll-like receptor 4 activator ligand MPLA, Toll-like receptor 9 ligand, MF59, oligodeoxynucleotide (CpG-ODN) and Freund's adjuvant.

[0027] The present invention includes the clinical use of the vaccine platform as a HBV therapeutic vaccine in combination with a hepatitis B virus envelope protein HBsAg vaccine in the treatment of chronic hepatitis B virus infection.

[0028] The present invention includes the clinical use of the vaccine platform as a HBV therapeutic vaccine in combination with nucleoside or nucleotide analogs in the treatment of chronic hepatitis B virus infection.

[0029] The present invention includes the combined application of the vaccine platform as a preventive or therapeutic vaccine for HBV, etc., with antiviral drugs and other treatment methods; and the combined application of a preventive or therapeutic vaccine for HBV-related tumors with antiviral and antitumor drugs and therapies.

[0030] The present invention comprises a multivalent combination vaccine consisting of the vaccine platform as a component of a vaccine and other viruses, pathogens or tumor vaccines.

[0031] The present invention comprises any one of the fusion protein vaccines of the vaccine platform and an adenovirus vaccine, mRNA vaccine, inactivated vaccine or DNA vaccine of the same virus for immunization in a sequential or simultaneous immunization program.

[0032] The present invention encompasses the full-length sequence and any truncated sequences of the vaccine platform antigen.

[0033] The present invention contains any possible mutants of the fusion protein vaccine antigen, including natural point mutations / deletion mutations / truncations, any combination of natural point mutations, subtypes produced after mutation, and artificial point mutations / deletion mutations / truncations and other mutation sequences constructed by the inventor of this patent to enhance the vaccine effect.

[0034] Any vaccine of the present invention as a component of a vaccine and another vaccine of the present invention or other vaccines different from the vaccine of the present invention, such as other viruses, pathogens or tumor vaccines, are used in a multivalent combination vaccine, for example, any vaccine of the present invention and adenovirus vaccine, mRNA vaccine, inactivated vaccine or DNA vaccine of the same virus are used for immunization in a sequential or simultaneous immunization program. As known in the art, when used in combination, the vaccines used in combination can be prepared as a kit for easy use.

[0035] Compared with the prior art, the present invention includes but is not limited to the following beneficial effects:

[0036] 1. The antigen of the IFN-virus antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention can be a variety of component changes, and can be a virus-specific antigen, which enhances the flexibility of the vaccine platform and also enhances the scope of use of this vaccine platform.

[0037] 2. The IFN-viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention, wherein the interferon (IFN) can enhance the migration and maturation of antigen-presenting cells, increase the costimulatory molecules expressed by them, thereby making them more conducive to presenting antigens to T cells. At the same time, the Fc region (or antibody) in the vaccine platform, on the one hand, enhances the molecular weight of the antigen and thus increases its half-life, and on the other hand, the Fc region (or antibody) can bind to the Fc receptors on the surface of antigen-presenting cells to promote the processing and presentation of antigens by antigen-presenting cells, thereby being more conducive to the generation of immune responses.

[0038] 3. The IFN-viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention is expressed by a eukaryotic HEK293 cell expression system. The protein expressed by HEK293 cells is closer to the natural protein molecule in terms of molecular structure, physicochemical characteristics, protein modification and biological function of the protein.

[0039] 4. The IFN-viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention has two structures of homologous or heterologous dimers, which has better selection for different antigens.

[0040] 5. The IFN-viral antigen-immunoglobulin Fc vaccine platform provided by the present invention can activate DCs to enhance DC cross-presentation and generate powerful B cell and T cell immune responses by fusing Th cell helper epitopes, such as Pan epitopes, using DC targeting antibodies such as anti-PD-L1, and adding various adjuvants that stimulate immune responses.

[0041] 6. The IFN-viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention has a wide range of uses and can be used not only as a preventive vaccine but also as a therapeutic vaccine.

[0042] 7. The IFN-viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention can be used not only alone, but also in combination with existing commercial HBsAg vaccines and nucleoside / nucleotide analogs as a therapeutic vaccine.

[0043] 8. The vaccine platform provided by the present invention can be used as a component of a vaccine to form a multivalent combination vaccine with other viruses, pathogens or tumor vaccines.

[0044] 9. Any fusion protein vaccine in the vaccine platform provided by the present invention can be used for immunization with adenovirus vaccine, mRNA vaccine, inactivated vaccine or DNA vaccine of the same virus in a sequential or simultaneous immunization schedule.

[0045] 10. The full-length sequence and any truncated sequence of the vaccine platform antigen provided by the present invention.

[0046] 11. Any possible mutants of the vaccine platform antigen provided by the present invention, including natural point mutations / deletion mutations / addition mutations / truncations, any combination of natural point mutations, subtypes produced after mutation, and artificial point mutations / deletion mutations / addition mutations / truncations and other mutation sequences constructed by the inventor of this patent to enhance the vaccine effect.

[0047] Sequence information involved in this article:

[0048] 1. Unit component sequence:

[0049] SEQ ID NO.1: Mouse mIFNα4 amino acid sequence (mIFNα)

[0050] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKE

[0051] SEQ ID NO.11: Human IFNα2 amino acid sequence (hIFNα)

[0052] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE

[0053] SEQ ID.NO.12: Amino acid sequence of human mutant IFNα2 (Q124R) (hmIFNα)

[0054] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE

[0055] SEQ ID NO.2: Amino acid sequence of human IgG1-Fc

[0056] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV

[0057] SEQ ID No.13: Heterodimeric Fc-hole

[0058] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0059] SEQ ID No.14: Heterodimer Fc-knob

[0060] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0061] SEQ ID NO.3: Th helper epitope Pan HLA DR-binding epitope (PADER) amino acid sequence

[0062] AKFVAAWTLKAAA

[0063] SEQ ID NO.4: Linker 1 amino acid sequence:

[0064] GGGGSGGGGSGGGGS

[0065] SEQ ID NO.15: Linker 2 amino acid sequence:

[0066] GSGSGS

[0067] SEQ ID NO.5: Signal peptide amino acid sequence:

[0068] MARLCAFLMILVMMSYYWSACSLG

[0069] SEQ ID NO.6: Amino acid sequence of HBV Pres1 (ad subtype)

[0070] MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVSTIPPPASTNRQSGRQPTPISPPLRDSHPQA

[0071] SEQ ID NO.16: Amino acid sequence of HBV Pres1 (ay subtype)

[0072] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQA

[0073] SEQ ID NO.7: Amino acid sequence of HBV HBsAg (adr subtype)

[0074] MENTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYNILSPFLPLLPIFFCLWVYI

[0075] SEQ ID NO.17: Amino acid sequence of HBV HBsAg (adw subtype)

[0076] MENITSGLLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLSFLGEAPVCLGQNSQSPTRNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMIWYWGPSLYSIVCPFTPLLQIFCCLWVFI

[0077] SEQ ID NO.18: Amino acid sequence of HBV HBsAg (ayw subtype)

[0078] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQSSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI

[0079] 2. Murine IFN vaccine mIFNα - antigen - Fc sequence:

[0080] SEQ ID NO.8: Amino acid sequence of mIFNα - Pres1 - Fc in a homodimer

[0081] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV

[0082] SEQ ID NO.9: Amino acid sequence of the first chain mIFNα-Fc-hole in the heterodimer

[0083] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0084] SEQ ID NO.10: Amino acid sequence of the second chain Pres1-Fc-knob in the heterodimer mIFNα-Pres1-Fc

[0085] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0086] 3. Mouse IFN vaccine containing Pan epitope IFNα-Pan-antigen-Fc sequence:

[0087] SEQ ID NO.19: Amino acid sequence of mIFNα-Pan-Pres1-Fc in the homodimer

[0088] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKEGGGGSGGGGSGGGGSRTAKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0089] SEQ ID NO.20: Amino acid sequence of the first chain mIFNα-Fc-hole in the heterodimer

[0090] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0091] SEQ ID NO.21: Amino acid sequence of the second chain Pan-Pres1-Fc-knob in the heterodimer mIFN-Pan-Pres1-Fc

[0092] AKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0093] 4. Human IFN vaccine hIFNα-antigen-Fc sequence:

[0094] SEQ ID NO.22: Amino acid sequence of hIFNα-Pres1-Fc in the homodimer

[0095] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0096] SEQ ID NO.23: Amino acid sequence of the first chain hIFN-Fc-hole in the heterodimer

[0097] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFKLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0098] SEQ ID NO.24: Amino acid sequence of the second chain Pres1-Fc-knob in the heterodimer hIFNα-Pres1-Fc

[0099] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0100] 5. Human IFN-containing Pan epitope vaccine IFNα-Pan-antigen-Fc sequence:

[0101] SEQ ID NO.25: Amino acid sequence of hIFNα-Pan-Pres1-Fc in the homodimer

[0102] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0103] SEQ ID NO.26: Amino acid sequence of the first chain hIFNα-Fc-hole in the heterodimer

[0104] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0105] SEQ ID NO.27: Amino acid sequence of the second chain Pan-Pres1-Fc-knob in the heterodimer hIFNα-Pan-Pres1-Fc

[0106] AKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0107] 6. Human mutant IFN vaccine hmIFNα-Pan-antigen-Fc sequence:

[0108] SEQ ID NO.28: Amino acid sequence of hmIFNα-Pres1-Fc in homodimer

[0109] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLY LKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTL PANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0110] SEQ ID NO.29: Amino acid sequence of the first chain hmIFN-Fc-hole in the heterodimer

[0111] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGV GVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0112] SEQ ID NO.30: Amino acid sequence of the second chain Pres1-Fc-knob in the heterodimer hmIFNα-Pres1-Fc

[0113] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0114] 7. Human mutant IFN containing Pan epitope vaccine hmIFNα-Pan epitope-antigen-Fc sequence

[0115] SEQ ID NO.31: Amino acid sequence of hmIFNα-Pan-Pres1-Fc in homodimer

[0116] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0117] SEQ ID NO.32: Amino acid sequence of the first chain hmIFNα4-Fc-hole in the heterodimer

[0118] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGV GVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0119] SEQ ID NO.33: Amino acid sequence of the second chain Pan-Pres1-Fc-knob in the heterodimer hmIFNα-Pan-Pres1-Fc

[0120] AKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1 . Schematic diagram of the vaccine platform in the form of a homodimer, arranged in the order of interferon-linking fragment 1-target antigen-immunoglobulin Fc (or antibody);

[0122] Figure 2 . The vaccine platform is in the form of a heterodimer, with a schematic diagram of the combination of interferon-linker fragment 1-IgG1-hole and target antigen-IgG1-knob (or antibody);

[0123] Figure 3 . The vaccine platform is in the form of a heterodimer, according to the schematic diagram of the interferon-linker fragment 1-IgG1-knob combination and the target protein-IgG1-hole (or antibody);

[0124] Figure 4 . Schematic diagram of the vaccine platform in the form of a homodimer, arranged in the order of interferon-linking fragment 1-Th cell helper epitope-linking fragment 2-target antigen-immunoglobulin Fc (or antibody);

[0125] Figure 5 . Schematic diagram of the vaccine platform in the form of heterodimers, respectively according to the combination of interferon-linking fragment 1-IgG1-hole and Th cell helper epitope-linking fragment 2-target antigen-IgG1-knob (or antibody);

[0126] Figure 6 . Schematic diagram of the vaccine platform in the form of heterodimers, respectively according to the combination of interferon-linking fragment 1-IgG1-knob and Th cell helper epitope-linking fragment 2-target antigen-IgG1-hole (or antibody).

[0127] Figure 7 . SDS-PAGE electrophoresis identification of Pres1-Fc and IFN-Pres1-Fc non-denatured proteins

[0128] Figure 8 Compared with free preS1, fusion proteins preS1-Fc and IFN-preS1-Fc can significantly enhance the immunogenicity of antigen molecules and induce the production of broad-spectrum neutralizing antibodies. (a) C57 / BL6 mice (n=8 / group) were subcutaneously immunized with free HBV Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins, and the levels of Pres1-specific antibodies in serum were detected by Elisa at the specified time. (b) Mice (n=4) stably carrying the three HBV genotypes were injected intravenously with serum from mice immunized with IFNα-Pres1-Fc protein, and the changes in Pres1 antigen in serum were detected 12 hours later.

[0129] Fig. 9IFNα-Pres1-Fc can be used as a preventive vaccine for hepatitis B. C57 / BL6 mice were subcutaneously immunized with free hepatitis B Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins. On day 28 of vaccination, 1x10 11 vg of AAV-HBV1.3 virus. (a) Serum Anti-Pres1 levels before virus inoculation and at 1, 2, 3, and 4 weeks after virus inoculation. (b) Serum Pres1 levels were detected at designated time points. (c) Serum HBsAg levels were detected at 1, 2, 3, and 4 weeks by Elisa. (d) The proportion of HBsAg-positive mice after AAV-HBV1.3 virus inoculation.

[0130] Fig.10 IFNα-Pres1-Fc as a therapeutic vaccine for chronic hepatitis B infection. C57 / BL6 mice were infected with 1x10 11 vg of AAV-HBV1.3 virus, and stably infected mice were selected after 6 weeks of infection (n=8 / group), and recombinant Pres1 and IFNα-Pres1-Fc proteins were inoculated subcutaneously, once every 2 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 HBV-related antigen HBsAg level in mouse serum

[0131] Fig.11 Th cell helper epitopes enhance antibody responses to IFNα-Pres1-Fc vaccine

[0132] Compared with IFN-preS1-Fc, IFN-Pan-preS1-Fc can significantly enhance the immunogenicity of the antigen molecule. C57 / BL6 (n=8 / group) mice were subcutaneously immunized with hepatitis B Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins without aluminum adjuvant, and the level of Pres1-specific antibodies in serum was detected by Elisa at the specified time.

[0133] Fig.12 IFNα-Pan-Pres1-Fc as a therapeutic vaccine for chronic hepatitis B infection. C57 / BL6 mice were infected with 1x10 11vg of AAV-HBV1.3 virus, and stably infected mice (n=8 / group) were selected after 6 weeks of infection. Recombinant Pres1 and IFNα-Pres1-Fc proteins were inoculated subcutaneously, and immunized once every 2 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-related antigen HBsAg level in mouse serum; (d) Detection of HBV-DNA level in mouse serum by QPCR.

[0134] Fig.13 IFNα-Pres1-Fc combined with HBsAg commercial vaccine breaks the immune tolerance caused by HBsAg and induces HBsAg-HBsAb serological conversion. HBV Carrier mice were subcutaneously immunized with IFNα-Pres1-Fc and HBsAg commercial vaccine, once every two weeks for a total of three immunizations. (a) The level of Pres1 in the serum of HBV Carrier mice, (b) the level of HBsAg, (c) the level of Anti-Pres1 in the serum, (d) the level of Anti-HBsAg in the serum, and (e) the level of HBV-DNA in the serum. ***, p<0.001 DETAILED DESCRIPTION

[0135] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be described in detail with reference to the embodiments and drawings below. The embodiments introduced are only examples of the present invention, but are not intended to limit the scope of the present invention. The examples are only a part of the present invention, but not all embodiments. The scope of the present invention is limited by the specific requirements of the attached claims.

[0136] Example 1. Design of vaccine platform

[0137] The vaccine platform of interferon-target antigen-immunoglobulin Fc (or antibody) structural unit is composed of three structural units. The first structural unit is the interferon part, the second structural unit is the immunoglobulin Fc region (or antibody), and the third unit is the target antigen. In actual construction, the three structural units can be arranged and combined in any form and the target antigen can be connected to the Th cell helper epitope through the connecting sequence 2. Its representative form is as follows:

[0138] Figure 1 . Schematic diagram of the vaccine platform in the form of a homodimer, arranged in the order of interferon-linking fragment 1-target antigen-immunoglobulin Fc.

[0139] Figure 2. Schematic diagram of the vaccine platform in the form of a heterodimer, respectively combining interferon-linking fragment 1-IgG1-hole and target antigen-IgG1-knob.

[0140] Figure 3 . Schematic diagram of the vaccine platform in the form of a heterodimer, according to the combination of interferon-linking fragment 1-IgG1-knob and target protein-IgG1-hole.

[0141] Next, we connect the target antigen to the cell helper epitope through the linker fragment 2, and then combine it with the other two vaccine platform components, which are represented as follows:

[0142] Figure 4 . A schematic diagram of the vaccine platform in the form of a homodimer, arranged in the order of interferon-linking fragment 1-Th cell helper epitope-linking fragment 2-target antigen-immunoglobulin Fc.

[0143] Figure 5 . Schematic diagram of the vaccine platform in the form of a heterodimer, respectively according to the combination of interferon-linking fragment 1-IgG1-hole and Th cell helper epitope-linking fragment 2-target antigen-IgG1-knob.

[0144] Figure 6 . Schematic diagram of the vaccine platform in the form of a heterodimer, respectively according to the combination of interferon-linking fragment 1-IgG1-knob and Th cell helper epitope-linking fragment 2-target antigen-IgG1-hole.

[0145] Example 2. Construction, purification and production of vaccine platform

[0146] We describe the expression and production of this vaccine platform using the homodimer form of hepatitis B virus Pres1 protein as an example.

[0147] 1. Vector construction, host cell transfection and induced expression

[0148] 1.1. Using PEE12.4 as a vector, the vaccine structural unit was constructed into the vector by molecular cloning to obtain a plasmid that can express the fusion protein. The fusion protein was then transiently transfected into 293F cells, the culture supernatant was collected, and the target protein was finally purified by Protein A affinity chromatography.

[0149] Vector construction (taking HBV preS1 antigen as an example)

[0150] (1) PEE12.4-HindIII-Signal Peptide 1-IFN-BsiwI-Pres1-BstbI-hIgG1-EcoRI

[0151] (2) PEE12.4-HindIII-Signal Peptide 1-Interferon-Bsiwi-PADER-Pres1-hIgG1-EcoRI

[0152] The connecting sequence between each fusion protein fragment is

[0153] (1) There is a linker fragment 1 between interferon and Pres1

[0154] (2) The linker sequence between interferon and PADER is linker fragment 1, and the linker fragment between PADER and Pres1 is linker fragment 2

[0155] 1.2. Transient transfection to quickly express the target protein:

[0156] (1) Cell recovery: Freestyle 293F cells were cultured at 3×10 7 Cryopreserved in CD OptiCHOTM media (containing 10% DMSO) at a concentration of 10 cells / ml. After being taken out of liquid nitrogen, the cells were quickly thawed in a 37°C water bath, added to a 15ml centrifuge tube containing 10ml OptiCHOTM media, and centrifuged at 1,000rpm for 5min. The supernatant was discarded, and the cell pellet was suspended and cultured in 30ml OptiCHOTM media at 37°C, 8% CO 2 , 135rpm. After 4 days, the cells were expanded and the concentration should not exceed 3×10 6 cells / ml.

[0157] (2) Two days before transfection, prepare suspension cultured 293F cells for transient transfection (200 ml) at a seeding density of 0.6-0.8×10 6 cells / ml.

[0158] (3) Two days later, count the transfected cell suspension and estimate the cell density to be 2.5-3.5×10 6 cells / ml, then centrifuge the cell suspension at 1,000 rpm for 5 min and discard the supernatant.

[0159] (4) Resuspend the cells in 50 ml of fresh Freestyle 293 media, centrifuge again at 1,000 rpm for 5 min, and discard the supernatant.

[0160] (5) Resuspend the 293F cells in 200 ml Freestyle 293 media.

[0161] (6) Dilute 600 μg of plasmid with 5 ml of Freestyle 293 media and sterilize by filtration using a 0.22 μM filter.

[0162] (7) Dilute 1.8 mg of PEI with 5 ml of Freestyle 293 media and sterilize by filtration using a 0.22 μM filter. Immediately mix 5 ml of plasmid and 5 ml of PEI and let stand at room temperature for 5 minutes.

[0163] (8) Add the plasmid / PEI mixture to the cell suspension and place at 37°C, 8% CO 2 , cultured in an 85rpm incubator, and supplemented with growth factor 50ug / L LONG™ R3IGF-1.

[0164] (9) After 4 hours, add 200 ml of EX-CELLTM 293 media and 2 mM Glutamine and adjust the rotation speed to 135 rpm to continue culturing.

[0165] (10) After 24 hours, add 3.8 mM VPA, a cell proliferation inhibitor. After 72 hours, add 40 ml of medium D and continue culturing. After 6-8 days (cell viability is less than 70%), collect the supernatant for the next purification step.

[0166] 1.3. Collection, purification and electrophoresis verification of fusion protein

[0167] 2. Purify the target protein using Protein A:

[0168] (1) Sample preparation: Transfer the suspended cell culture medium to a 500 ml centrifuge bucket and centrifuge at 8,000 rpm for 20 min. Discard the precipitate, filter the supernatant through a 0.45 μM filter to remove impurities, and then add NaN to a final concentration of 0.05%. 3 Prevent bacterial contamination during purification.

[0169] (2) Assemble the chromatography column: Take an appropriate amount of Protein A Agarose (calculated as 20 mg human Fc fusion protein per 1 ml of Protein A), mix well and add to the chromatography column. Let it stand at room temperature for about 10 min. After the Protein A and 20% ethanol solution are separated, open the outlet at the bottom to allow the ethanol solution to slowly flow out by gravity.

[0170] (3) Rinse and equilibrate the column with 10 column volumes of distilled water and Binding buffer (20 mM sodium phosphate + 0.15 M NaCl, pH 7.0), respectively.

[0171] (4) Load the sample using a constant flow pump at a flow rate of 10 column volumes per hour, collect the flow-through, and repeat the loading twice.

[0172] (5) Rinse the column with 10 times the column volume of Binding Buffer to remove impurities and continue washing until no protein is detected in the effluent.

[0173] (6) Use Elution Buffer (0.1 M Glycine, pH 2.7) for elution. Collect the eluate in separate tubes, collecting one tube for every 1 ml, and observe the elution peak using a protein indicator solution (Bio-Rad protein assay). Mix the collection tubes of the elution peak and add an appropriate amount of 1 M Tris, pH 9.0 to neutralize (adjust the pH to 6-8, which should be at least 0.5 different from the isoelectric point of the purified protein).

[0174] (7) Use Zeba desalting centrifugal columns or concentrating centrifugal columns to replace the target protein solution into the required buffer (note to adjust the buffer pH to avoid the isoelectric point of the protein). Use BSA as the standard and determine the protein concentration by SDS-PAGE electrophoresis and NanoDrop2000.

[0175] (8) After elution, rinse the column with 20 times the column volume of distilled water, then rinse the column with 10 times the column volume of 20% ethanol. Finally, the ethanol solution should submerge the gel medium and store at 4°C.

[0176] 3. The protein SDS-PAGE electrophoresis identification diagram is as follows Figure 7 shown.

[0177] Example 3. IFNα-Pres1-Fc and Pres1-Fc can induce stronger immune responses in mice than simple Pres1 antigen.

[0178] Materials: C57BL / 6 male mice (5-8 weeks) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Beijing Kangwei Biotechnology Co., Ltd.; 96-well ELISA assay plates were purchased from Corning Costar; ELISA colorimetric solution was purchased from eBioscience; the microplate reader SPECTRA max PLUS 384 was purchased from Molecular Corporation, USA. The aluminum adjuvant used was purchased from SIGMA.

[0179] method:

[0180] (1) Immunization of mice with Pres1 fusion protein: 80 pmol IFN-Pres1-Fc or 80 pmol Pres1-Fc, Pres1 protein and aluminum adjuvant were mixed and subcutaneously immunized into mice. The serum of mice was collected by orbital bleeding at the designated time points for antibody detection.

[0181] (2) The antibodies produced by IFNα-Pres1-Fc have a broad neutralizing effect on different genotypes of HBV. 5-week-old male C57BL / 6 mice were infected with AAV-HBV 1.3 (HBV genotypes B, C, and D) via the tail vein. 1x10 11 vg virus, and 6 weeks later, mice with continuous and stable expression of HBV antigen were selected for experiments. The selected mice (4 mice / group) were injected intravenously with 200μl / mouse of serum from IFNα-Pres1-Fc immunized mice. After 12 hours, the mouse serum was collected, and the changes of Pres1 antigen in the mice before and after the injection of antiserum were detected by Elisa.

[0182] (3) ELISA detection of anti-Pres1 specific antibodies in serum. Add 50 μl of Pres1 (2 μg / ml) coating solution to the Elisa plate (Corning 9018) in each well and coat overnight at 4°C. Wash once with PBS, 260 μl per well. Block with 5% blocking solution (5% FBS) at 37°C for two hours. Dilute serum samples with PBS (1:10, 1:100, 1:1000, 1:10000), add 50 μl to each well of the blocked Elisa plate and incubate at 37°C for 1 hour. Wash 5 times with PBST, 260 μl each time, add 50 μl of enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS) to each well, and incubate at 37°C for 1 hour. Wash five times with PBST, 260μl each time, add 100μl / well of substrate TMB, incubate at room temperature in the dark, and wait for the substrate to develop color; add 50μl stop solution (2N H2SO4) to each well to stop color development, read the plate with an ELISA reader, OD450-630.

[0183] Results: The immunogenicity of free Pres1 was weak. When IFNα and Fc were added to Pres1 to form IFNα-Pres1-Fc fusion protein, its immunogenicity was greatly improved. Figure 8 (a) As shown. The antibodies induced by IFNα-Pres1-Fc can produce a wide range of neutralizing effects on different HBV genotypes, such as Figure 8 (b) shown.

[0184] Example 4. IFNα-Pres1-Fc can be used as a preventive vaccine against hepatitis B

[0185] Materials: C57BL / 6 (6-8) week old male mice were purchased from Beijing Weitonglihua Biotechnology Co., Ltd., and HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd. AAV-HBV 1.3 virus was purchased from Guangzhou Paizhen Biotechnology Co., Ltd. Other experimental materials were the same as in Example 3.

[0186] method:

[0187] (1) Mice were subcutaneously immunized with 80 pmol of different forms of Pres1 vaccine, including simple Pres1, Pes1-Fc, and IFNα-Pres1-Fc protein. On day 28 of immunization, mouse sera were collected and mice were infected with 1x10 11 vgAAV-HBV 1.3 virus, and then collect mouse serum every week to test anti-Pres1 antibodies, HBsAg, and Pres1 antigen in the serum for four consecutive weeks. The peripheral HBV-DNA level of mice was tested in the third week.

[0188] (2) ELISA detection of Pres1-specific antigen in serum. Antigen coating: Add 50 μl of the coating solution of Pres1 antibody XY007 (4 μg / ml) to the Elisa plate (Corning 9018) per well and coat overnight at 4°C. Wash once with PBS, 260 μl per well. Block with 5% blocking solution (5% FBS) at 37°C for two hours. Dilute the serum sample with PBS (1:10, 1:100), add 50 μl to each well of the blocked Elisa plate, set up two replicate wells for each dilution, and incubate at 37°C for 1 hour. Wash 5 times with PBST, 260 μl each time, add 50 μl of enzyme conjugate (from Kehua HBsAg Detection Kit) to each well, and incubate at 37°C for 1 hour. Wash five times with PBST, 260 μl each time, add 100 μl / well of substrate TMB, incubate at room temperature in the dark, and wait for the substrate to develop color; add 50 μl of stop solution (2N H 2 SO 4 ) Stop color development and read the plate with an ELISA reader, OD450-630.

[0189] Results: The mice in the IFNα-Pres1-Fc immunization group were able to produce high levels of Pres1 antibodies before virus inoculation, and the antibodies continued to maintain a high level during the virus infection. Fig. 9 (a). Compared with the group without protein immunization, IFN-Pres1-Fc vaccine immunization can significantly prevent HBV infection, and the anti-preS1 antibodies produced after immunization can quickly and completely eliminate preS1 antigens in the serum Fig. 9 (b) In addition, most of the mice infected with the virus in the IFN-Pres1-Fc immunization group showed peripheral HBsAg negative Fig. 9 (c, d). The above experimental results show that IFN-Pres1-Fc as a vaccine can effectively prevent HBV infection. Fig. 9 shown.

[0190] Example 5. IFNα-Pres1-Fc as a therapeutic vaccine for chronic influenza B infection

[0191] Materials: C57BL / 6 male mice (4 weeks old) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. AAV-HBV 1.3 was purchased from Guangzhou Paizhen Biotechnology Co., Ltd. HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd. Other experimental materials were the same as in Example 4.

[0192] method:

[0193] (1) Screening of HBV Carrier Mice: 4-week-old HBV C57BL / 6 mice were injected with 1x10 11 vg AAV-HBV 1.3 virus, HBV antigen HBsAg was detected in 1-6 weeks, and mice with stable HBsAg expression were screened out and used as HBV Carrier mice for experiments.

[0194] (2) The selected mice were injected subcutaneously with 80 pmol of different forms of Pres1 protein, once every two weeks for a total of three immunizations. Mouse serum was collected on day 14 of immunization and then once a week. The levels of anti-Pres1 antibodies, HBsAg, and Pres1 antigen in the mouse serum were tested by ELISA. The HBV-DNA content in the mouse peripheral blood was tested after the last blood draw.

[0195] Results: We detected the changes in serum preS1 antigen, serum Pres1 antibody and HBsAg in Carrier mice after immunization with IFNα-Pres1-Fc vaccine. The results showed that after IFNα-Pres1-Fc vaccine immunization, mice produced high levels of anti-Pres1 antibodies. Fig.10 (a), and the preS1 antigen in the serum can be completely cleared, as shown in Fig.10 (b) As shown, HBsAg in serum also decreased to a certain extent as shown in Figure 10(c), while the untreated control group and the group immunized with Pres1 vaccine alone had no therapeutic effect. Fig.10 shown.

[0196] Example 6. T cell helper epitopes enhance the antibody response to IFNα-Pres1-Fc vaccine

[0197] Materials: Same as Example 3

[0198] method:

[0199] (1) Immunize mice with Pres1 fusion protein. Immunize mice subcutaneously with 80 pmol of IFN-Pan-Pres1-Fc containing the Pan epitope or 80 pmol of IFN-Pan-Pres1-Fc, Pres1-Fc, and Pres1 protein. Collect serum from mice by orbital bleeding at designated time points for antibody detection.

[0200] (2) ELISA was used to detect anti-Pres1 specific antibodies in serum, as in Example 3.

[0201] Results: Compared with fusion protein vaccines such as IFN-preS1-Fc, IFN-Pan-preS1-Fc can significantly enhance the immunogenicity of antigen molecules and induce the production of broad-spectrum neutralizing antibodies. C57 / BL6 (n=8 / group) mice were subcutaneously immunized with hepatitis B Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins without aluminum adjuvant, and the level of Pres1-specific antibodies in serum was detected by Elisa at the designated time.

[0202] Example 7. IFNα-Pan-Pres1-Fc as a therapeutic vaccine for chronic influenza B infection

[0203] Materials: C57BL / 6 male mice (4 weeks old) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. AAV-HBV 1.3 was purchased from Guangzhou Paizhen Biotechnology Co., Ltd. HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd. Other experimental materials were the same as in Example 4.

[0204] method:

[0205] (1) Screening of HBV Carrier Mice: 4-week-old HBV C57BL / 6 mice were injected with 1x10 11 vg AAV-HBV 1.3 virus, HBV antigen HBsAg was detected in 1-6 weeks, and mice with stable HBsAg expression were screened out and used as HBV Carrier mice for experiments.

[0206] (2) The selected mice were injected subcutaneously with 80 pmol of different forms of Pres1 protein, once every two weeks for a total of three immunizations. Mouse serum was collected on day 14 of immunization, and then collected once a week. The levels of anti-Pres1 antibodies, HBsAG, and Pres1 antigen in the mouse serum were tested by ELISA. The HBV-DNA content in the mouse peripheral blood was tested after the last blood draw.

[0207] Results: We detected the changes in serum preS1 antigen, serum Pres1 antibody and HBsAg in Carrier mice after immunization with IFN-Pan-Pres1-Fc vaccine. The results showed that after immunization with IFN-Pan-Pres1-Fc vaccine, mice produced high levels of anti-Pres1 antibodies such as Fig.12 (a) As shown. And accompanied by the complete removal of preS1 antigen in serum Fig.12 (b) As shown, the HBsAg in the serum also decreased to a certain extent12(c), while the untreated control group and the group immunized with Pres1 vaccine alone had no therapeutic effect. In addition, the HBV DNA also decreased significantly in the IFNα-Pan-Pres1-Fc immunization group. Fig.12 (d) as shown.

[0208] Example 8. IFNα-Pan-Pres1-Fc combined with HBsAg commercial vaccine breaks the immune tolerance caused by HBsAg and induces HBsAg-HBsAb serological conversion.

[0209] Materials: C57BL / 6 male mice (4 weeks old) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. AAV-HBV 1.3 was purchased from Guangzhou Paizhen Biotechnology Co., Ltd. HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd., and Anti-HBsAg kit was purchased from Beijing Wantai Biopharmaceutical Co., Ltd. Commercial HBsAg vaccine was purchased from Aimi Hanxin Vaccine (Dalian) Co., Ltd. Other experimental materials were the same as in Example 7.

[0210] method:

[0211] (1) Screening of HBV Carrier Mice: 4-week-old HBV C57BL / 6 mice were injected with 1x10 11 vg AAV-HBV 1.3 virus, HBV antigen HBsAg was detected in 1-6 weeks, and mice with stable HBsAg expression were screened out and used as HBV Carrier mice for experiments.

[0212] (2) The HBV Carrier mice screened were immunized with 80 pmol IFNα-pan-Pres1-Fc and 2 μg of commercial HBsAg vaccine twice, with an interval of 14 days between each immunization. Mouse serum was collected 14 days after the first immunization, and then weekly, and the changes in anti-Pres1, Pres1, anti-HBsAg, and HBsAg in the serum were detected. The level of HBV-DNA in the serum was detected at the last mouse serum collection.

[0213] Results: We found that the combination of IFNα-Pan-Pres1-Fc and commercial HBsAg as a strategy for treating chronic hepatitis B can ultimately break HBsAg tolerance. The immune response generated in HBV-tolerant mice can completely eliminate preS1 antigen in the serum Fig.13 (a), and there are high concentrations of Pres1 antibodies in the serum13 (c). Excitingly, the IFN-Pan-Pres1-Fc vaccine effectively cleared HBsAg in the serum and induced some serological HBsAb conversion Fig.13 (b) and (d), which are considered to be the key indicators of HBV cure in clinical practice. In addition, we detected the expression level of HBV-related DNA in peripheral blood by fluorescence quantitative PCR (real-time PCR). The results showed that compared with the control group, the immunization method of IFNα-Pan-Pres1-Fc combined with commercial HBsAg can ultimately reduce the level of peripheral HBV DNA. Fig.13 (e) Based on the above results, we invented a vaccine strategy for treating chronic hepatitis B by combining IFNα-Pan-Pres1-Fc and commercial HBsAg vaccine.

[0214] References:

[0215] [1] WHO W. Global hepatitis report 2017 [J]. Geneva: World HealthOrganization, 2017,

[0216] [2] RAZAVI-SHEARER D, GAMKRELIDZE I, NGUYEN M H, et al. Globalprevalence, treatment, and prevention of hepatitis B virus infection in 2016:a modelling study [J]. The lancet Gastroenterology & hepatology, 2018, 3(6):383-403.

[0217] [3] SCHWEITZER A, HORN J, MIKOLAJCZYK R T, et al. Estimations ofworldwide prevalence of chronic hepatitis B virus infection: a systematicreview of data published between 1965 and 2013 [J]. The Lancet, 2015, 386(10003): 1546-55.

[0218] [4] VOS T, ABAJOBIR A A, ABATE K H, et al. Global, regional, andnational incidence, prevalence, and years lived with disability for 328diseases and injuries for 195 countries, 1990–2016: a systematic analysis forthe Global Burden of Disease Study 2016 [J]. The Lancet, 2017, 390(10100):1211-59.

[0219] [5] KONTERMANN R E. Strategies for extended serum half-life ofprotein therapeutics [J]. Current opinion in biotechnology, 2011, 22(6): 868-76.

[0220] [6] MEKHAIEL D N, CZAJKOWSKY D M, ANDERSEN J T, et al. Polymerichuman Fc-fusion proteins with modified effector functions [J]. Scientificreports, 2011, 1(1): 1-11.

[0221] 7] ROOPENIAN D C, AKILESH S. FcRn: the neonatal Fc receptor comes ofage [J]. Nature reviews immunology, 2007, 7(9): 715-25.

[0222] [8] VILCEK J. Fifty years of interferon research: aiming at a movingtarget [J]. Immunity, 2006, 25(3): 343-8.

[0223] [9] BRACCI L, LA SORSA V, BELARDELLI F, et al. Type I interferons asvaccine adjuvants against infectious diseases and cancer [J]. Expert reviewof vaccines, 2008, 7(3): 373-81.

[0224]

[10] HAHM B, TRIFILO M J, ZUNIGA E I, et al. Viruses evade the immunesystem through type I interferon-mediated STAT2-dependent, but STAT1-independent, signaling [J]. Immunity, 2005, 22(2): 247-57.

[0225]

[11] ITO T, AMAKAWA R, INABA M, et al. Differential regulation ofhuman blood dendritic cell subsets by IFNs [J]. The Journal of Immunology,2001, 166(5): 2961-9.

[0226]

[12] MONTOYA M, SCHIAVONI G, MATTEI F, et al. Type I interferonsproduced by dendritic cells promote their phenotypic and functionalactivation [J]. Blood, 2002, 99(9): 3263-71.

[0227]

[13] LE BON A, ETCHART N, ROSSMANN C, et al. Cross-priming of CD8+ Tcells stimulated by virus-induced type I interferon [J]. Nature immunology,2003, 4(10): 1009-15.

[0228]

[14] LE BON A, DURAND V, KAMPHUIS E, et al. Direct stimulation of Tcells by type I IFN enhances the CD8+ T cell response during cross-priming[J]. The Journal of Immunology, 2006, 176(8): 4682-9.

[0229]

[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 AmericanSociety of Hematology, 2012, 119(6): 1407-17.

[0230]

[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 dendriticcells: importance for the rapid acquisition of potent migratory andfunctional activities [J]. Blood, The Journal of the American Society ofHematology, 2001, 98(10): 3022-9.

[0231]

[17] ROUZAUT A, GARASA S, TEIJEIRA Á, et al. Dendritic cells adhereto and transmigrate across lymphatic endothelium in response to IFN‐α [J].European journal of immunology, 2010, 40(11): 3054-63.

[0232]

[18] LE BON A, SCHIAVONI G, D'AGOSTINO G, et al. Type I interferonspotently enhance humoral immunity and can promote isotype switching bystimulating dendritic cells in vivo [J]. Immunity, 2001, 14(4): 461-70。

Claims

1. A vaccine comprising a fusion protein containing interferon, a target antigen, and an immunoglobulin Fc region as structural units, The interferon is the first structural unit, as shown in the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 11, or SEQ ID NO: 12, The target antigen is the third structural unit, which is HBV Pres1 antigen. wherein the immunoglobulin Fc region is the second structural unit, The fusion protein also comprises one or more Th cell helper epitopes and a connecting fragment.

2. The vaccine according to claim 1, wherein the fusion protein is a homodimeric fusion protein, the homodimeric fusion protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain and the second polypeptide chain are completely identical, and the first polypeptide chain and the second polypeptide chain comprise interferon, Th cell helper epitope, target antigen and immunoglobulin Fc region in sequence from N-terminus to C-terminus.

3. The vaccine according to claim 1 or 2, wherein the immunoglobulin Fc region is the Fc region of IgG1, IgG2, IgG3, IgG4 or IgM, preferably the Fc region of IgG1.

4. The vaccine according to any one of claims 1 to 3, wherein the target antigen is the HBV Pres1 antigen shown in 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. The vaccine according to any one of claims 1-4, wherein the Th cell helper epitope amino acid sequence is shown as SEQ ID NO:

3.

6. The vaccine according to any one of claims 1-5, wherein the connecting fragments between the structural units of the fusion protein are flexible polypeptide sequences, as shown in the amino acid sequences of SEQ ID NO: 4 and / or SEQ ID NO:

15.

7. Use of the fusion protein in the vaccine according to any one of claims 1 to 6 in the preparation of a composition or kit for use as a preventive or therapeutic vaccine for hepatitis B.

8. The use according to claim 7, wherein the composition or kit is used for the prevention or treatment of HBV, such as the composition or kit is used as a preventive or therapeutic vaccine for hepatitis B.

9. The vaccine according to any one of 1 to 6 or the use according to claim 7 or 8, It is characterized in that The vaccine, the composition or the kit can be administered via intramuscular, intravenous, transdermal, subcutaneous or nasal immunization routes, wherein the vaccine, the composition or the kit can also include an adjuvant, and the adjuvant can include aluminum adjuvant (Alum), Toll-like receptor 4 activator ligand MPLA, Toll-like receptor 9 ligand, oligodeoxynucleotide (CpG-ODN), MF59 and Freund's adjuvant.

10. The vaccine according to any one of claims 1 to 6 or the use according to any one of claims 7 to 9, It is characterized in that The vaccine can be used in combination with other preventive or therapeutic therapies, for example, the vaccine can be a hepatitis B therapeutic vaccine, the hepatitis B therapeutic vaccine can be used in combination with other preventive or therapeutic hepatitis B therapies, 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 combined with nucleoside or nucleotide analogs, for example, for the treatment of chronic hepatitis B virus infection, for example, the vaccine is a component of a vaccine in a multivalent combination vaccine composed of other viruses or pathogens or tumor vaccines, for example, the vaccine is immunized with an adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of the same virus in a sequential or simultaneous immunization program.

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