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 fields of genetic engineering and biomedical technology, specifically relating to a vaccine, such as a fusion protein vaccine comprising an interferon-target antigen-immunoglobulin Fc region (antibody) as its primary framework. The vaccine of the present invention can be used as a vaccine platform to prevent hepatitis B virus (HBV) infection, HPV, EBV, HIV, SARA-CoV-2, influenza virus infection, and HPV- and EBV-related tumors, as well as to treat chronic hepatitis B (CHB) infection and HBV-, HPV-, and EBV-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. HBV causes approximately 30% of liver cirrhosis and 40% of hepatocellular carcinoma (HCC). . Hepatitis B virus infection remains a major global public health problem. 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; and long-term medication causes large 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 is of very important social and economic significance.

[0004] Seasonal influenza causes severe illness in 1-4 million people and 200,000-500,000 deaths each year. . Vaccines are the best way to prevent and control influenza. Vaccines can reduce the incidence of disease and the severity of infection, especially in young children and the elderly, who are at risk of influenza complications. Even though the currently approved influenza vaccines can produce good protection in healthy young adults, there are still some problems that need to be solved. For example, the production of some vaccines relies on chicken embryos, such as inactivated influenza vaccines and attenuated influenza vaccines. One disadvantage of these vaccines is that if the prevalent strains are of avian origin, the epidemic of the disease will lead to an increase in demand for vaccines and chicken embryos, which will cause problems with the supply of chicken embryos. Another disadvantage is that these vaccines take a long time to produce. Older people are more likely to develop severe symptoms from influenza viruses, and standard vaccines are generally less effective in older people, whose immune systems weaken with age. In view of the problems encountered by current influenza vaccines, there is an urgent need for an influenza vaccine that has strong immunogenicity, does not rely on chicken embryos, and can be produced quickly to prevent the spread of influenza viruses.

[0005] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen that causes the 2019 coronavirus disease (COVID-19) pandemic. The clinical symptoms caused by SARS-CoV-2 mainly include asymptomatic infection, mild flu-like symptoms, pneumonia and severe acute respiratory distress syndrome. In severe cases, it can cause death in infected patients. There is currently no specific drug for the new coronavirus, and vaccines are the basic countermeasures to control and end the new coronavirus pandemic. In addition, the emergence of new coronavirus mutants has posed new challenges to existing vaccine candidates and the control of the epidemic. Therefore, a strong vaccine that can also work against the new coronavirus mutants is urgently needed under the current epidemic situation.

[0006] When the antigen is linked to the immunoglobulin Fc region, the half-life of the antigen will be significantly extended, and the immunoglobulin Fc region can bind to the Fc receptor on the surface of the antigen-presenting cell, thereby promoting the processing and presentation of the antigen by the antigen-presenting cell. Type I interferon, as an antiviral cytokine, has many biological activities, one of which includes the stimulation of immune cells. IFNα can strongly induce the differentiation and activation of human DC cells After acting on immature DCs, type I interferon 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. It has been reported that type I interferon can promote the antigen presentation ability of DCs after infection with vaccinia virus and lymphocytic chorioencephalitis virus (LCMV). In addition, type I interferon can promote the migration of DCs to lymph nodes by upregulating the expression of chemokine receptors after acting on DCs, thereby promoting the activation of T cells. . Recently, more and more studies have shown that type I interferon can be used as an immune adjuvant. Le Bon et al.'s study showed that when mice were immunized with weak immunogens, type I interferon showed a strong immune adjuvant effect in mice, inducing the production of long-lasting antibodies and immune memory. The authors also found that DCs are the primary cell population where type I interferon exerts its effects. Using antibodies to deliver the vaccine to DCs, stimulating DC activation and cross-presentation, will further enhance the activity and potency of the vaccine.

[0007] The present invention provides a need for a vaccine platform that enhances the body's response to viral, bacterial or tumor antigens. Summary of the Invention

[0008] Vaccines are an effective way to prevent and control major infectious diseases. There are various types of vaccines, an important category of which is protein subunit vaccines. Generally speaking, the immunogenicity of simple protein subunit vaccines is generally poor, which often limits the use of protein subunit vaccines. Therefore, a universal protein subunit vaccine platform is urgently needed. Based on the effects of the immunoglobulin Fc region and type I interferon on the immune system, the inventors have specially proposed an interferon α-virus antigen, bacteria or tumor-immunoglobulin Fc region fusion protein vaccine platform to enhance the body's response to viruses, bacteria or tumor antigens. The present invention provides a type I interferon-protein antigen-immunoglobulin Fc vaccine platform, in which type I interferon can act on antigen-presenting cells to make them mature and migrate, thereby better playing the role of antigen presentation and activation of T cells. On the other hand, the Fc part of the vaccine platform can bind to the Fc receptor on the surface of antigen-presenting cells to enhance the uptake of antigens by antigen-presenting cells, thereby further helping antigen-presenting cells to play their role. The inventors have proposed that the fusion of Th cell helper epitopes can further enhance the immune response of the type I interferon-protein antigen-immunoglobulin Fc vaccine, making it a key component of the vaccine. The inventors have proposed that antibodies such as anti-PD-L1 could be used to replace the Fc, targeting the vaccine to DCs, stimulating DC activation and cross-presentation, further enhancing the activity and potency of the vaccine. This invention, as a novel vaccine platform, can be used as a preventive and therapeutic vaccine for diseases such as viral infections, bacterial infections, and tumors.

[0009] In some embodiments, the present invention provides a vaccine comprising an interferon-target antigen-immunoglobulin Fc region (or antibody) fusion protein (with attached Th epitopes). In some embodiments, the present invention also provides the use of an interferon-target antigen-immunoglobulin Fc region (or antibody) fusion protein (with attached Th epitopes) for the preparation of a prophylactic or therapeutic composition or kit (e.g., a pharmaceutical or vaccine composition or kit). The vaccine of the present invention can be produced using a eukaryotic cell expression system and administered via subcutaneous / intramuscular or intranasal routes of immunization. The antibody (Ab) serving as the structural unit of the fusion polypeptide of the present invention is not particularly limited and may include, for example, a complete antibody or antibody fragment, such as an antibody heavy and light chain, or a single-chain antibody. It may be a DC-targeting activating antibody, including anti-PD-L1, anti-DEC205, anti-CD80 / 86, and other antibodies.

[0010] 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 tumor antigen and / or a pathogen antigen (such as a viral or bacterial antigen). In some embodiments, the target antigen described herein can be, for example, a tumor antigen, such as a protein molecule highly expressed on tumor cells, for example, human epidermal growth factor receptor 2 (HER2 / neu) or epidermal growth factor (EGFR).

[0011] In some embodiments, the target antigen used in the vaccine provided by the present invention can be, for example, a mutated target antigen that is different from the wild type. In some embodiments, the target antigen described herein can be, for example, a mutant of a tumor antigen and / or a pathogen antigen (such as a viral or bacterial antigen). In some embodiments, the target antigen can be, for example, the full length or S1 region of the SARS-COV-2 viral S protein, for example, the target antigen can be the antigen set forth in SEQ ID NO.76 or SEQ ID NO.77. In this article, 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 the majority allele in nature and is often used as a standard control gene in biological experiments), such as the Spike protein (S protein) derived from the original wild-type strain of SARS-CoV-2. In this article, the mutated target antigen (mutant) refers to the mutated viral protein expressed by the mutant virus strain encoded by the mutant gene derived from the wild-type gene. For example, the point mutations of the S protein of the new coronavirus that have been discovered include: NTD region 69-70 deletion, Y144 deletion, 242-244 deletion, L18F, D80A, D215, R246I mutations, RBD region K417, E484, N501Y and other mutations, L452R mutation, T478K mutation, D614G, H655Y mutations. For example, these point mutations exist in different combinations in novel coronaviruses derived from the UK B.1.1.7 (Alpha) mutant, South Africa B.1.351 (Beta) mutant, Brazil P1 (Gamma) mutant, India B.1.617, B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.617.3 mutant, California B.1.429 mutant, etc. In some embodiments, the mutated target antigen may include, for example, natural point mutations / deletion mutations / addition mutations / truncations, artificial point mutations / deletion mutations / addition mutations / truncations, any combination of natural or artificial mutations, and subtypes produced by mutations, wherein the target antigen may be a tumor antigen, a pathogen antigen, such as a virus (e.g., SARS-COV-2), or a bacterial antigen.In some embodiments, the target antigen used in the vaccine provided by the present invention is a mutated viral antigen, for example, the mutated viral antigen can be a mutant of SARS-COV-2, including, for example, natural point mutations / deletion mutations / addition mutations / truncations of SARS-COV-2 proteins (for example, one or more of S protein, N protein, M protein, and E protein), artificial point mutations / deletion mutations / addition mutations / truncations, any combination of natural or artificial mutations, and subtypes produced after mutation. For example, the mutated viral antigen can be a mutant of the full-length S protein, S1 region, or RBD region, for example The mutated viral antigen may comprise one or more of the following mutations of the S protein of SARS-COV-2: NTD region 69-70 deletion, Y144 deletion, 242-244 deletion, L18F, D80A, D215, R246I mutations, RBD region K417, E484, N501Y mutations, L452R mutations, T478K mutations, D614G, H655Y mutations, for example, the mutated viral antigen may comprise a B.1.1.7 (Alpha) mutant strain from the UK, a B.1.351 (Beta) mutant from South Africa, or a B.1.1.7 (Alpha) mutant strain from the UK. Mutant strains and mutations in the Brazilian P1 (Gamma) mutant strain, Indian B.1.617, B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.617.3 mutant strain, and California B.1.429 mutant strain, for example, the mutated viral antigen may comprise a mutant of any one of SEQ ID NO.79, SEQ ID NO.80, and SEQ ID NO.81, for example, the mutated viral antigen may be a mutant comprising any one of SEQ ID NO.79, SEQ ID NO.80, SEQ ID NO.81, SEQ ID NO.82, SEQ ID NO.83, and SEQ ID NO.84. Herein, unless otherwise expressly stated or clearly limited by the context, the target antigens mentioned herein generally include wild-type target antigens and mutant target antigens.

[0012] The present invention provides a vaccine platform comprising interferon (IFN) and tumor, bacterial or viral antigens (hepatitis B virus Pres1 antigen, SARS-COV2 RBD antigen, influenza HA antigen, human papillomavirus HPVE7 antigen, hepatitis B virus surface antigen (HBsAg) or peptide, herpes zoster virus (VZV) gE antigen, Epstein-Barr virus (EBV) EBNA1 / LMP2 / gp350, herpes simplex virus 2 (HSV-2) gD The fusion protein may be a homologous or heterologous dimeric protein. When the fusion protein is a dimer, the interferon, target antigen, and immunoglobulin Fc region (or antibody) as structural units may be present in the first polypeptide chain and / or the second polypeptide chain. The presence of each structural unit is not particularly limited. For example, they may be present simultaneously in one chain, or any one or more structural units may be present in one chain while another one or more structural units are present in the other chain.

[0013] 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 of human or mouse origin; preferably type I interferon IFN-α (SEQ ID NO.1, SEQ ID NO.21, SEQ ID NO.22).

[0014] 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.23, SEQ ID NO.24).

[0015] The fusion polypeptides of the present invention may optionally further comprise one or more Th cell helper epitopes and / or a linker. For example, when the fusion protein is a dimer, the fusion protein may further comprise one or more Th cell helper epitopes and / or a linker in either one or both 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 an appropriate linker. The linker that can be used in the vaccines of the present invention is not particularly limited and may be any suitable peptide fragment known in the art. The linker between the various structural units of the present invention may be a flexible polypeptide sequence, such as linker 1 and linker 2, as shown, for example, in the amino acid sequences of SEQ ID NO. 4 and SEQ ID NO. 25.

[0016] The N-terminus of the polypeptide sequence composed of each structural unit of the present invention contains a corresponding signal peptide capable of promoting protein secretion, such as shown in the amino acid sequence of SEQ ID NO.5.

[0017] 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.26), HBV HBsAg antigen (various subtypes and peptides), including adr subtype (SEQ ID NO.7), adw subtype (SEQ ID NO.27), and ayw subtype (SEQ ID NO.28), SARA-COV2 RBD antigen (SEQ ID NO.8), influenza virus HA antigen (SEQ ID NO.9), HPV E7 antigen (SEQ ID NO.10), herpes simplex virus VZV-gE antigen (SEQ ID NO.91), EBV-gp350 antigen (SEQ ID NO.92), and HSV-2-gD antigen (SEQ ID NO.93).

[0018] The homodimeric protein described herein comprises a first polypeptide and a second polypeptide, the first polypeptide being identical to the second polypeptide. The first and second polypeptides, from N-segment to C-segment, consist of an IFN-tumor or viral antigen (hepatitis B Pres1 antigen, SARS-CoV-2 RBD antigen, influenza HA antigen, HPV E7 antigen, HBsAg antigen, VZV-gE antigen, EBV EBNA1 / LMP2 / gp350, HSV-2-gD antigen, HIV gp120 antigen)-immunoglobulin Fc region; or a polypeptide containing a Pan epitope. The amino acid sequences comprise SEQ ID NOs. 11, 12, 13, 14, 29, 30, 31, 32, 38, 39, 40, 47, 48, 49, 50, 51, 56, 57, 59, 58, 65, 66, 67, and 68.

[0019] The heterodimer of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different polypeptides. The first polypeptide comprises an IFN-immunoglobulin Fc region from the C segment to the N-terminus, comprising the amino acid sequences shown in SEQ ID NOs. 15, 33, 42, 51, 60, and 69; the second polypeptide comprises a tumor or viral antigen (hepatitis B Pres1 antigen, SARS-COV2 RBD antigen, influenza HA antigen, HPV E7 antigen, VZV-gE antigen, EBV EBNA1 / LMP2 / gp350, HSV-2-gD antigen, HIV gp120 antigen)-immunoglobulin Fc region from the C segment to the N-terminus; comprising SEQ ID NO. The amino acid sequences shown in 16, 17, 18, 19, 34, 35, 36, 37, 43, 44, 45, 46, 52, 53, 54, 55, 61, 62, 63, 64, 70, 71, 72, and 73.

[0020] The present invention also provides amino acid sequences encoding the above-mentioned IFN-tumor or viral antigen hepatitis B Pres1 antigen, HBsAg antigen or peptide, SARS-COV2 RBD antigen, influenza HA antigen, HPV E7 antigen, VZV-gE antigen, EBV EBNA1 / LMP2 / gp350, HSV-2-gD antigen, HIV gp120 antigen-immunoglobulin Fc vaccine platform.

[0021] The present invention also relates to nucleotide fragments encoding the vaccine platform and fusion protein.

[0022] 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:

[0023] (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;

[0024] (2) constructing a host cell containing the expression vector by transiently transfecting the host cell. Preferably, the host cell is a 293F cell;

[0025] (3) culturing the host cells and collecting the cell supernatant;

[0026] (4) Purify the fusion protein or vaccine platform by protein A / G affinity chromatography column purification.

[0027] The present invention further includes the use of the vaccine platform, which can be used as a preventive vaccine for hepatitis B, the use of the vaccine platform as a therapeutic vaccine for hepatitis B, the use of the vaccine platform as a preventive vaccine for influenza, the use of the vaccine platform as a preventive vaccine for SARA-COV2, influenza, HPV, VZV, EBV, HSV-2, and HIV, and the use of the vaccine platform as a preventive vaccine for HPV and EBV-related tumors.

[0028] The present invention includes adjuvants used in the vaccine platform, wherein the adjuvants include aluminum adjuvant (Alum), Toll-like receptor 4 activator ligand MPLA, Toll-like receptor 9 ligand, MF59, oligodeoxynucleotide (CpG-ODN) and Freund's adjuvant.

[0029] The present invention includes the clinical use of the vaccine platform as an HBV therapeutic vaccine in combination with a hepatitis B virus envelope protein HBsAg vaccine in the treatment of chronic hepatitis B virus infection.

[0030] The present invention includes the clinical use of the vaccine platform as an HBV therapeutic vaccine in combination with nucleoside or nucleotide analogs in the treatment of chronic hepatitis B virus infection.

[0031] The present invention includes the combined use of the vaccine platform as a preventive or therapeutic vaccine for HBV, influenza, SARA-COV2, HPV, VZV, EBV, HSV-2, HIV, etc., in combination with antiviral drugs and other treatment methods; and the combined use of preventive or therapeutic vaccines for HBV, HPV, EBV-related tumors in combination with antiviral and anti-tumor drugs and therapies.

[0032] 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.

[0033] 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, pathogen or tumor for immunization in a sequential or simultaneous immunization program.

[0034] The present invention includes the full-length sequence and any truncated sequence of the vaccine platform antigen, such as SEQ ID NO.76, SEQ ID NO.77, and SEQ ID NO.78.

[0035] Any possible mutants of the fusion protein vaccine antigen contained in the present invention include natural point mutations / deletion mutations / truncations, any combination of natural point mutations, subtypes produced after mutation, and artificial point mutations / deletion mutations / truncations constructed by the inventor of this patent to enhance the vaccine effect, such as SEQ ID NO.79, SEQ ID NO.80, SEQ ID NO.81, SEQ ID NO.82, SEQ ID NO.83, and SEQ ID NO.84.

[0036] A multivalent combination vaccine composed of 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 virus or pathogen or tumor vaccines, for example, a multivalent vaccine composed of the SARS-COV-2 fusion protein vaccine of the present invention and an influenza vaccine or other vaccine, for example, any vaccine of the present invention and an adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of the same virus, pathogen or tumor are immunized in a sequential or simultaneous immunization program, for example, a SARS-COV-2 fusion protein vaccine and an adenovirus vaccine or mRNA vaccine or inactivated vaccine of SARS-COV-2 Immunization with a SARS-CoV-2 fusion protein vaccine or DNA vaccine can be performed sequentially or simultaneously. For example, the sequential immunization sequence can be: 1) first immunization with the SARS-CoV-2 fusion protein vaccine of the present invention, followed by immunization with adenovirus vaccine, mRNA vaccine, inactivated vaccine, or DNA vaccine for SARS-CoV-2; 2) first immunization with adenovirus vaccine, mRNA vaccine, inactivated vaccine, or DNA vaccine for SARS-CoV-2, followed by immunization with the SARS-CoV-2 fusion protein vaccine; 3) simultaneous immunization with adenovirus vaccine, mRNA vaccine, inactivated vaccine, or DNA vaccine for SARS-CoV-2. As known in the art, when used in combination, the combined vaccines can be prepared as a convenient kit.

[0037] Compared with the prior art, the present invention includes but is not limited to the following beneficial effects:

[0038] 1. The IFN-tumor or viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention can have antigens of various components, including both tumor-associated antigens and virus-specific antigens. This enhances the flexibility and scope of application of the vaccine platform.

[0039] 2. The IFN-tumor or viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention contains interferon (IFN) that can enhance the migration and maturation of antigen-presenting cells, thereby increasing the co-stimulatory molecules they express, 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, thereby increasing 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, thereby promoting the processing and presentation of the antigen by the antigen-presenting cells, thereby more conducive to the generation of an immune response.

[0040] 3. The IFN-tumor or viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention is expressed in a eukaryotic HEK293 cell expression system. The proteins expressed by HEK293 cells are closer to natural protein molecules in terms of molecular structure, physicochemical characteristics, protein modification, and biological function.

[0041] 4. The IFN-tumor or viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention has two structures: homologous or heterologous dimers, which provide better selection for different antigens.

[0042] 5. The IFN-tumor or viral antigen-immunoglobulin Fc vaccine platform provided by the present invention can activate DCs and enhance DC cross-presentation 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, thereby generating powerful B cell and T cell immune responses.

[0043] 6. The IFN-tumor or 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.

[0044] 7. The IFN-tumor or 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.

[0045] 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.

[0046] 9. Any fusion protein vaccine in the vaccine platform provided by the present invention can be used for immunization with an adenovirus vaccine, mRNA vaccine, inactivated vaccine, or DNA vaccine for the same virus, pathogen, or tumor in a sequential or simultaneous immunization schedule.

[0047] 10. The full-length sequence and any truncated sequence of the vaccine platform antigen provided by the present invention.

[0048] 11. Any possible mutants of the vaccine platform antigens provided by the present invention, including natural point mutations / deletion mutations / addition mutations / truncations, any combination of natural point mutations, subtypes produced by mutation, and artificial point mutations / deletion mutations / addition mutations / truncations constructed by the inventors of this patent to enhance the efficacy of the vaccine.

[0049] Sequence information involved in this article:

[0050] 1. Unit component sequence:

[0051] SEQ ID NO.1: Mouse mIFNα4 amino acid sequence (mIFNα)

[0052] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKE

[0053] SEQ ID NO.21: Human IFNα2 amino acid sequence (hIFNα)

[0054] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKE

[0055] SEQ ID. NO. 22: Human mutant IFNα2 (Q124R) amino acid sequence (hmIFNα)

[0056] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE

[0057] SEQ ID NO.2: Amino acid sequence of human IgG1-Fc

[0058] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV

[0059] SEQ ID No.23: Heterodimeric Fc-hole

[0060] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0061] SEQ ID No.24: Heterodimeric Fc-knob

[0062] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0063] SEQ ID NO.3: Th helper epitope Pan HLA DR-binding epitope (PADER) amino acid sequence

[0064] AKFVAAWTLKAAA

[0065] SEQ ID NO.4: Linker 1 amino acid sequence:

[0066] GGGGSGGGGSGGGGS

[0067] SEQ ID NO.25: Linker 2 amino acid sequence:

[0068] GSGSGS

[0069] SEQ ID NO.5: Signal peptide amino acid sequence:

[0070] MARLCAFLMILVMMSYYWSACSLG

[0071] SEQ ID NO.6: Amino acid sequence of HBV Pres1 (ad subtype)

[0072] MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVSTIPPPASTNRQSGRQPTPISPPLRDSHPQA

[0073] SEQ ID NO.26: Amino acid sequence of HBV Pres1 (ay subtype)

[0074] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQA

[0075] SEQ ID NO.7: Amino acid sequence of HBV HBsAg (adr subtype)

[0076] MENTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYNILSPFLPLLPIFFCLWVYI

[0077] SEQ ID NO.27: Amino acid sequence of HBV HBsAg (adw subtype)[[ID=,10]]

[0078] MENITSGLLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLSFLGEAPVCLGQNSQSPTRNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMIWYWGPSLYSIVCPFTPLLQIFCCLWVFI

[0079] SEQ ID NO.28: Amino acid sequence of HBV HBsAg (ayw subtype)

[0080] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQSSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI

[0081] SEQ ID NO.8: Amino acid sequence of SARS-CoV-2 RBD

[0082] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0083] SEQ ID NO.9: Amino acid sequence of HA of influenza virus

[0084] DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSR

[0085] SEQ ID NO.10: Amino acid sequence of HPV-E7 antigen

[0086] MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP

[0087] 2. Mouse IFN vaccine mIFNα - antigen - Fc sequence:

[0088] SEQ ID NO.11: Amino acid sequence of mIFNα - Pres1 - Fc in the homodimer

[0089] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV

[0090] SEQ ID NO.12: Amino acid sequence of mIFNα - RBD (SARS - CoV - 2) - Fc in the homodimer

[0091] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV

[0092] SEQ ID NO.13: Amino acid sequence of mIFNα-HA-Fc in the homodimer

[0093] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV

[0094] SEQ ID NO.14: Amino acid sequence of mIFNα-E7 (HPV)-Fc in a homodimer

[0095] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV

[0096] SEQ ID NO.15: Amino acid sequence of the first chain mIFNα-Fc-hole in the heterodimer

[0097] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0098] SEQ ID NO.16: Amino acid sequence of the second chain Pres1-Fc-knob in the heterodimeric mIFNα-Pres1-Fc

[0099] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0100] SEQ ID NO.17: Amino acid sequence of the second chain RBD (SARS-CoV-2)-Fc-knob in the heterodimeric mIFNα-RBD (SARS-CoV-2)-Fc

[0101] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0102] SEQ ID NO.18: Amino acid sequence of the second chain HA-Fc-knob in the heterodimeric mIFNα-HA-Fc

[0103] DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0104] SEQ ID NO.19: Amino acid sequence of the second chain E7-Fc-knob in the heterodimeric mIFNα-E7 (HPV)-Fc

[0105] MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0106] 3. Mouse IFN vaccine containing Pan epitope IFNα-Pan-antigen-Fc sequence:

[0107] SEQ ID NO.29: Amino acid sequence of mIFNα-Pan-Pres1-Fc in homodimer

[0108] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSL CAWEVIRAEVWRALSSSTNLLARLSEEKEGGGGSGGGGSGGGGSRTAKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQ AQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0109] SEQ ID NO.30: mIFNα-Pan-RBD (SARS-CoV-2)-Fc amino acid sequence in homodimer

[0110] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKEGGGGSGGGGSGGGGSRTAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0111] SEQ ID NO.31: Amino acid sequence of mIFNα-Pan-HA-Fc in the homodimer

[0112] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKEGGGGSGGGGSGGGGSRTAKFVAAWTLKAAAGSGSGSDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0113] SEQ ID NO.32: Amino acid sequence of mIFNα - Pan - E7 (HPV)-Fc in a homodimer

[0114] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKEGGGGSGGGGSGGGGSRTAKFVAAWTLKAAAGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0115] SEQ ID NO.33: Amino acid sequence of the first chain mIFNα-Fc-hole in the heterodimer

[0116] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0117] SEQ ID NO.34: Amino acid sequence of the second chain Pan-Pres1-Fc-knob in the heterodimeric mIFN-Pan-Pres1-Fc

[0118] AKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0119] SEQ ID NO.35: Amino acid sequence of the second chain Pan-RBD(SARS-CoV-2)-Fc-knob in the heterodimeric mIFNα-Pan-RBD (SARA-CoV-2)-Fc

[0120] AKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0121] SEQ ID NO.36: Amino acid sequence of the second chain Pan-HA-Fc-knob in the heterodimeric mIFNα-Pan-HA-Fc

[0122] AKFVAAWTLKAAAGSGSGSDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0123] SEQ ID NO.37: Amino acid sequence of the second chain Pan-E7-Fc-knob in the heterodimeric mIFNα-Pan-E7 (HPV)-Fc

[0124] AKFVAAWTLKAAAGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0125] 4. Human IFN vaccine hIFNα-antigen-Fc sequence:

[0126] SEQ ID NO.38: hIFNα-Pres1-Fc amino acid sequence in homodimer

[0127] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0128] SEQ ID NO.39: Amino acid sequence of hIFNα-RBD (SARS-CoV-2)-Fc in the homodimer

[0129] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0130] SEQ ID NO.40: Amino acid sequence of hIFNα-HA-Fc in the homodimer

[0131] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0132] SEQ ID NO.41: Amino acid sequence of hIFNα-E7 (HPV)-Fc in a homodimer,

[0133] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0134] SEQ ID NO.42: Amino acid sequence of the first chain hIFN-Fc-hole in the heterodimer

[0135] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFKLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0136] SEQ ID NO.43: Amino acid sequence of the second chain Pres1-Fc-knob in the heterodimer hIFNα-Pres1-Fc

[0137] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0138] SEQ ID NO.44: Amino acid sequence of the second chain RBD(SARS-CoV-2)-Fc-knob in the heterodimer hIFNα-RBD (SARA-CoV-2)-Fc

[0139] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0140] SEQ ID NO.45: Amino acid sequence of the second chain HA-Fc-knob in the heterodimer hIFNα-HA-Fc

[0141] DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0142] SEQ ID NO.46: Amino acid sequence of the second chain E7(HPV)-Fc-knob in the heterodimeric hIFNα-E7 (HPV)-Fc

[0143] MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0144] 5. Human IFN vaccine containing Pan epitope IFNα-Pan-antigen-Fc sequence:

[0145] SEQ ID NO.47: hIFNα-Pan-Pres1-Fc amino acid sequence in homodimer

[0146] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYL KEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQT LPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0147] SEQ ID NO.48: hIFNα-Pan-RBD (SARS-CoV-2)-Fc amino acid sequence in homodimer

[0148] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0149] SEQ ID NO.49: Amino acid sequence of hIFNα-Pan-HA-Fc in a homodimer

[0150] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0151] SEQ ID NO.50: Amino acids of hIFNα-Pan-E7 (HPV)-Fc in a homodimer

[0152] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0153] SEQ ID NO.51: Amino acid sequence of the first chain hIFNα-Fc-hole in the heterodimer

[0154] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0155] SEQ ID NO.52: Amino acid sequence of the second chain Pan-Pres1-Fc-knob in the heterodimer hIFNα-Pan-Pres1-Fc

[0156] AKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0157] SEQ ID NO.53: Amino acid sequence of the second chain Pan-RBD(SARS-CoV-2)-Fc-knob in the heterodimer hIFNα-Pan-RBD (SARA-CoV-2)-Fc

[0158] AKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0159] SEQ ID NO.54: Amino acid sequence of the second chain Pan-HA-Fc-knob in the heterodimer hIFNα-Pan-HA-Fc

[0160] AKFVAAWTLKAAAGSGSGSDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0161] SEQ ID NO.55: Amino acid sequence of the second chain, Pan-HA-Fc-knob, in the heterodimeric hIFNα-Pan-E7(HPV)-Fc

[0162] AKFVAAWTLKAAAGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0163] 6. Human mutant IFN vaccine hmIFNα-Pan-antigen-Fc sequence:

[0164] SEQ ID NO.56: Amino acid sequence of hmIFNα-Pres1-Fc in homodimer

[0165] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0166] SEQ ID NO.57: Amino acid sequence of hmIFNα-RBD (SARS-CoV-2)-Fc in a homodimer

[0167] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0168] SEQ ID NO.58: Amino acid sequence of hmIFNα-HA-Fc in a homodimer

[0169] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0170] SEQ ID NO.59: Amino acid sequence of hmIFNα-E7 (HPV)-Fc in a homodimer

[0171] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0172] SEQ ID NO.60: Amino acid sequence of the first chain hmIFN-Fc-hole in the heterodimer

[0173] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0174] SEQ ID NO.61: Amino acid sequence of the second chain Pres1-Fc-knob in the heterodimer hmIFNα-Pres1-Fc

[0175] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0176] SEQ ID NO.62: Amino acid sequence of the second chain RBD (SARS-CoV-2)-Fc-knob in the heterodimer hmIFNα-RBD (SARS-CoV-2)-Fc

[0177] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0178] SEQ ID NO.63: Amino acid sequence of the second chain HA-Fc-knob in the heterodimeric hmIFNα-HA-Fc

[0179] DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0180] SEQ ID NO.64: Amino acid sequence of the second chain HA-Fc-knob in the heterodimeric hmIFNα-E7 (HPV)-Fc

[0181] MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0182] 7. Human mutant IFN containing Pan epitope vaccine hmIFNα-Pan epitope-antigen-Fc sequence

[0183] SEQ ID NO.65: Amino acid sequence of hmIFNα-Pan-Pres1-Fc in homodimer

[0184] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYL KEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQT LPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0185] SEQ ID NO.66: Amino acid sequence of hmIFNα-Pan-RBD (SARS-CoV-2)-Fc in homodimer

[0186] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0187] SEQ ID NO.67: Amino acid sequence of hmIFNα-Pan-HA-Fc in a homodimer

[0188] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0189] SEQ ID NO.68: Amino acid sequence of hmIFNα-Pan-E7 (HPV)-Fc in a homodimer

[0190] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0191] SEQ ID NO.69: Amino acid sequence of the first chain hmIFNα4-Fc-hole in the heterodimer

[0192] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0193] SEQ ID NO.70: Amino acid sequence of the second chain Pan-Pres1-Fc-knob in the heterodimer hmIFNα-Pan-Pres1-Fc

[0194] AKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0195] SEQ ID NO.71: Amino acid sequence of the second chain Pan-RBD (SARS-CoV-2)-Fc-knob in the heterodimer hmIFNα-Pan-RBD (SARS-CoV-2)-Fc

[0196] AKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0197] SEQ ID NO.72: Amino acid sequence of the second chain Pan-HA-Fc-knob in the heterodimer hmIFNα-Pan-HA-Fc

[0198] AKFVAAWTLKAAAGSGSGSDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0199] SEQ ID NO.73: Amino acid sequence of the second chain Pan-HA-Fc-knob in the heterodimeric hmIFNα-Pan-E7 (HPV)-Fc

[0200] AKFVAAWTLKAAAGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0201] 8. Anti-Fc Antibody Sequences

[0202] SEQ ID NO.20: Amino Acid Sequence of ScFv(PD-L1)

[0203] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA

[0204] SEQ ID NO.74: Anti-PD-L1 VH Amino Acid Sequence

[0205] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA

[0206] SEQ ID NO.75: Anti-PD-L1 VL Amino Acid Sequence

[0207] DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR

[0208] 9. Other viral antigen sequences

[0209] SEQ ID NO. 76 Amino acid sequence of SARS-CoV-2 Spike protein

[0210]

[0211] Amino acid sequence of SARS-CoV-2 S1 protein of SEQ ID NO. 77

[0212] VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR

[0213] Amino acid sequence of RBD protein of the original strain of SARS-CoV-2 of SEQ ID NO. 78

[0214] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0215] SEQ ID NO. 79 Amino acid sequence of the RBD protein of the UK mutant strain of SARS-CoV-2 (B.1.1.7, Alpha)

[0216] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0217] SEQ ID NO. 80 Amino acid sequence of the RBD protein of the South African mutant strain of SARS-CoV-2 (B.1.351, Beta)

[0218] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0219] SEQ ID NO. 81 Amino acid sequence of the RBD protein of the Brazilian mutant strain (P.1) of SARS-CoV-2

[0220] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGTIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0221] SEQ ID NO. 82 Amino acid sequence of the RBD protein of the California mutant strain of SRAS-CoV-2 (B.1.429)

[0222] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0223] SEQ ID NO. 83 Amino acid sequence of RBD protein of SRAS-CoV-2 Indian B.1.617, B.1.617.1 (Kappa), and B.1.617.3 mutant strains

[0224] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVQGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0225] SEQ ID NO. 84 Amino acid sequence of the RBD protein of the second-generation SRAS-CoV-2 Indian B.1.617.2 (Delta) mutant strain

[0226] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0227] 10. Other tumor antigen sequences:

[0228] The amino acid sequences of the extracellular domains II, III, and IV of murine Her2 involved in the examples are:

[0229] SEQ ID NO. 85 Mouse Her2-extracellular domain 2:

[0230] SRACPPCAPACKDNHCWGESPEDCQILTGTICTSGCARCKGRLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMHNPEGRYTFGASCVTTCPYNYLSTEVGSCTLVCPPNNQEVTAEDGTQRCEKCSKPC

[0231] SEQ ID NO. 86 Mouse Her2-extracellular domain 3:

[0232] GCKKIFGSLAFLPESFDGDPSSGIAPLRPEQLQVFETLEEITGYLYISAWPDSLRRDLSVFQNLRIIRGRILHDGAYSLTLQGLGIHSLGLRSLRELGSGLALIHRNAHLCFVHTVPWDQLF

[0233] SEQ ID NO. 87 Mouse Her2-extracellular domain 4:

[0234] VCNSLCAHGHCWGPGPTQCVNCSHFLRGQECVEECRVWKGLPREYVSDKRCLPCHPECQPQNSSETCFGSEADQCAACAHYKDSSSCVARCPSGVKPDLSYMPIWKYPDEEGICQPCPINCTHSCVDLDERGCP

[0235] The amino acid sequences of the human Her2 extracellular domains II, III, and IV involved in the examples are:

[0236] SEQ ID NO. 88 Human Her2-extracellular domain 2:

[0237] SRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPC

[0238] SEQ ID NO. 89 Human Her2-extracellular domain 3:

[0239] GCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPW

[0240] SEQ ID NO. 90 Human Her2-extracellular domain 4:

[0241] CHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCP

[0242] 11. Herpes virus antigen sequences involved in the examples:

[0243] SEQ ID NO. 91 VZV Envelope glycoprotein E (aa 31-538)

[0244] SVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRY

[0245] SEQ ID NO. 92 EBV Envelope glycoprotein GP350 (aa 1-425)

[0246] MEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCTADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALTM RSKKLPINVTTGEEQQVSLESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNCNSTNITAVVRAQGLDVTLPLSLPTSAQDSNFSVKTQMLGNEIDIECI MEDGEISQVLPGDNKFNITCSGYESHVPSGGILTSTSPVVTPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKASGGDYCIQSNIVFSDEIPASQDMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWAWPNNTETDFKCKWTLTSGTPSGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTTHKVIFSKAP

[0247] SEQ ID NO. 93 HSV-2 Envelope glycoprotein gD (aa 26-339)

[0248] KYALADPSLKMADPNRFRGKNLPVLDRLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPA FETAGTYLRLVKINDWTEITQFILEHRRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNP BRIEF DESCRIPTION OF THE DRAWINGS

[0249] 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);

[0250] Figure 2Schematic diagram of the vaccine platform in the form of a heterodimer, with the interferon-linker fragment 1-IgG1-hole and the target antigen-IgG1-knob (or antibody) combination;

[0251] Figure 3 The vaccine platform is in the form of a heterodimer, with the interferon-linking fragment 1-IgG1-knob combination and the target protein-IgG1-hole (or antibody) combination as shown in the following diagram;

[0252] 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);

[0253] Figure 5 Schematic diagram of the vaccine platform in the form of heterodimers, with interferon-linking fragment 1-IgG1-hole and Th cell helper epitope-linking fragment 2-target antigen-IgG1-knob (or antibody) combinations respectively;

[0254] Figure 6 . Schematic diagram of the vaccine platform in the form of heterodimers, with the combination of interferon-linking fragment 1-IgG1-knob and Th cell helper epitope-linking fragment 2-target antigen-IgG1-hole (or antibody).

[0255] Figure 7 . SDS-PAGE electrophoresis identification of Pres1-Fc and IFN-Pres1-Fc non-denatured proteins

[0256] Figure 8 Compared to free preS1, the fusion proteins preS1-Fc and IFN-preS1-Fc significantly enhance the immunogenicity of the antigen and elicit the production of broadly neutralizing antibodies. (a) C57 / BL6 mice (n=8 / group) were subcutaneously immunized with free HBV Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins. Serum levels of Pres1-specific antibodies were measured using ELISA at the indicated times. (b) Mice (n=4) stably carrying the three HBV genotypes were intravenously injected with serum from mice immunized with IFNα-Pres1-Fc protein. Changes in serum Pres1 antigen were measured 12 hours later.

[0257] Figure 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. 28 days after vaccination, 1x10 11 AAV-HBV1.3 virus was injected into the vg. (a) Serum Anti-Pres1 levels before and at weeks 1, 2, 3, and 4 after virus inoculation. (b) Serum Pres1 levels were measured at the indicated time points. (c) Serum HBsAg levels were measured by ELISA at weeks 1, 2, 3, and 4. (d) Proportion of HBsAg-positive mice after AAV-HBV1.3 inoculation.

[0258] Figure 10 IFNα-Pres1-Fc was used as a therapeutic vaccine for chronic HBV infection. C57 / BL6 mice were infected with 1x10 11 vg of AAV-HBV1.3 virus. Six weeks after infection, mice with stable infection were selected (n=8 / group) and subcutaneously immunized with recombinant Pres1 and IFNα-Pres1-Fc proteins, with immunizations 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 HBV-associated antigen HBsAg levels in mouse serum

[0259] Figure 11 Th cell helper epitopes enhance antibody responses to IFNα-Pres1-Fc vaccines

[0260] Compared with IFN-preS1-Fc, IFN-Pan-preS1-Fc significantly enhanced the immunogenicity of the antigen molecule. C57 / BL6 mice (n=8 / group) were subcutaneously immunized with HBV Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins without aluminum adjuvant. Serum Pres1-specific antibody levels were measured using ELISA at designated times.

[0261] Figure 12 IFNα-Pan-Pres1-Fc as a therapeutic vaccine for chronic HBV infection. C57 / BL6 mice were infected with 1x10 11Mice were infected with AAV-HBV1.3 virus (vg) for 6 weeks. Stably infected mice (n=8 / group) were selected and subcutaneously immunized with recombinant Pres1 and IFNα-Pres1-Fc proteins, with immunizations occurring 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 HBV-associated antigen HBsAg levels in mouse serum; (d) Detection of HBV-DNA levels in mouse serum by QPCR.

[0262] Figure 13 IFNα-Pres1-Fc combined with a commercial HBsAg vaccine breaks immune tolerance against HBsAg and induces HBsAg-to-HBsAb seroconversion. 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) Serum Pres1 levels in HBV carrier mice, (b) HBsAg levels, (c) serum Anti-Pres1 levels, (d) serum Anti-HBsAg levels, and (e) serum HBV-DNA levels. ***, p < 0.001

[0263] Figure 14 IFNα-RBD (SARS-CoV2)-Fc elicits a stronger antibody response than free SARS-CoV-2 RBD protein. Balb / c mice (n=8 / group) were subcutaneously immunized with free SARS-CoV-2 RBD, RBD-Fc, and IFNα-RBD-Fc proteins. Serum levels of SARS-CoV-2 S protein-specific antibodies were measured using an ELISA at the indicated times. ****, p<0.0001.

[0264] Figure 15 IFNα-RBD (SARS-CoV2)-Fc immunized mice and produced high-titer antiviral serum, which could completely block SARS-CoV2 pseudovirus infection in in vitro cell experiments.

[0265] Figure 16Detection of anti-RBD-specific antibodies in serum produced by immunization with IFNα-Pan-RBD (original strain)-Fc and IFNα-Pan-RBD (SARS-CoV-2 South Africa mutant)-Fc. (a) SDS-PAGE electrophoresis of IFNα-Pan-RBD (SARS-CoV-2 original strain)-Fc. (b) SDS-PAGE electrophoresis of IFNα-Pan-RBD (SARS-CoV-2 South Africa mutant)-Fc. (c) Binding of RBD-specific antibodies to the original strain RBD in serum 14 days after immunization with IFNα-Pan-RBD (original strain)-Fc and IFNα-Pan-RBD (SARS-CoV-2 South Africa mutant)-Fc. (d) Binding of RBD-specific antibodies to the South African mutant RBD in serum 14 days after immunization with IFNα-Pan-RBD (original strain)-Fc and IFNα-Pan-RBD (SARS-CoV-2 South African mutant)-Fc.

[0266] Figure 17 (a) SDS-PAGE electrophoresis analysis of Mouse IFNα-RBD-Fc and Mouse IFNα-Pan-RBD-Fc proteins. (b) SDS-PAGE electrophoresis analysis of Human IFNα-RBD-Fc and Human IFNα-Pan-RBD-Fc proteins.

[0267] Figure 18 Pan (Pan DR-binding epitope) CD4 T cell helper epitopes can further enhance the immunogenicity of IFNα-RBD-Fc. Mice were immunized intramuscularly with 10 μg of mouse IFNα-RBD-Fc, mouse IFNα-Pan-RBD-Fc, or 10 μg of human IFNα-RBD-Fc, or human IFNα-Pan-RBD-Fc. A booster immunization was administered 14 days after vaccination. Serum was collected on days 7, 14, and 28 after immunization, and RBD-specific antibody levels were measured by ELISA. *, p<0.05; ****, p<0.0001.

[0268] Figure 19Aluminum adjuvants can enhance the specific humoral immune responses induced by human IFNα-RBD-Fc and human IFNα-Pan-RBD-Fc proteins. C57BL / 6 mice were vaccinated with 10 μg of human IFNα-RBD-Fc or human IFNα-Pan-RBD-Fc protein on days 0 and 14, either with (AL+) or without (AL-) the addition of aluminum adjuvant. Serum was collected on days 7, 14, and 28 after vaccination, and SARS-CoV-2 RBD-specific antibody levels in the serum were assessed by ELISA. *, p<0.05; ****, p<0.0001.

[0269] Figure 20 Intranasal immunization with IFN-Pan-RBD-Fc induces high titers of RBD-specific IgG and IgA neutralizing antibodies. Six- to eight-week-old C57BL / 6 mice were divided into five groups of 10 mice each and immunized with 10 μg of IFNα-pan-RBD-Fc or the same molar concentrations of RBD, RBD-Fc, or IFNα-RBD-Fc protein via intranasal immunization at a volume of 10 μL per mouse. Mice were immunized twice, on days 0 and 14. Sera were collected on days 7, 14, 21, 28, 35, and 42 after immunization, and ELISA was used to assess the levels of IgG (a) and IgA (b) specific for the SARS-CoV-2 RBD in each serum group. Sera collected on day 42 were used for in vitro neutralization of SARS-CoV-2 pseudoviruses (c). Statistical method: one-way ANOVA, *p<0.05 indicates significant difference, **p<0.01, ***p<0.001, ****p<0.0001.

[0270] Figure 21 Intranasal immunization with the unadjuvanted COVID-19 vaccine IFN-Pan-RBD-Fc induced high titers of RBD-specific IgG and IgA neutralizing antibodies in the nasopharynx and lung tissues. Six- to eight-week-old C57BL / 6 mice were sacrificed 28 days after immunization, and the nasal mucosa was harvested and homogenized using a tissue homogenizer. The homogenized fluid was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was obtained as the nasal mucosal supernatant (NMDS). ELISA was used to assess the levels of IgG (a) and IgA (b) specific for COVID-19 RBD in the NMDS of each group. Serum collected 28 days after immunization was used for in vitro neutralization of SARS-CoV-2 pseudoviruses (c). Statistical analysis: One-way ANOVA. *P < 0.05 indicates a significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0271] Figure 22Intranasal immunization with the unadjuvanted COVID-19 vaccine IFN-Pan-RBD-Fc induced high titers of RBD-specific IgG and IgA antibodies in lung tissue. 6-8 week old C57BL / 6 mice 28 days after immunization (e.g. Figure 19 ) were killed. For the mouse lungs, about 0.8 ml of HBSS + 100uM EDTA was drawn into the tracheal tube with a 1 ml syringe, and the solution was gently blown and aspirated three times. The liquid was then aspirated and collected in a centrifuge tube. This step was repeated three times, and about 2 ml of lung lavage fluid was finally obtained. The mouse lung lavage fluid was centrifuged at 500 g for 5 minutes. The supernatant was the mouse lung lavage fluid (BALF). The ELISA method was used to detect the content of IgG (a) and IgA (b) specific antibodies to the new coronavirus RBD in the lung lavage fluid (BALF) of each group of mice. Statistical method: one-way ANOVA, *p<0.05 showed significant differences, **p<0.01, ***p<0.001, ****p<0.0001.

[0272] Figure 23 Her2 vaccine protein expression and purification. The relevant proteins were expressed and purified in 293F cells. Protein size and purity were assessed by SDS-PAGE and Coomassie blue staining. a. IFNα-3-Fc (62.6 kDa); b. IFNα-pan-3-Fc (63.9 kDa); c. IFNα-pan-4-Fc (74.9 kDa) and IFNα-4-Fc (73.6 kDa).

[0273] Figure 24 The antitumor activity of the Her2 vaccines IFNα-3-Fc and IFNα-pan-3-Fc was analyzed. TUBO breast cancer models were established in mice with tumors ranging from 50 to 80 mm³. The relevant fusion proteins were injected intratumorally once weekly for three treatments. The IFNα-3-Fc dose was 10 μg / dose / mouse, and the other fusion proteins were administered in equal moles. CpG was used as an adjuvant. Tumor size was measured, and tumor growth curves were plotted.

[0274] Figure 25 To analyze the ability of IFNα and Pan to enhance the immunogenicity of the Her2 antigen. Six- to eight-week-old BALB / C mice (n=5) were subcutaneously immunized with the mouse Her2 fusion protein vaccines 4-Fc, IFNα-4-Fc, and IFNα-pan-4-Fc, without adjuvant. The immunization dose was 10 μg / dose of IFNα-4-Fc per mouse, and the other fusion proteins were administered at equal molar amounts. Venous blood was collected 14 and 21 days after immunization, and serum levels of Her2-specific IgG were measured by ELISA.

[0275] Figure 26. SDS-PAGE electrophoresis identification pattern of IFN-HA1-Fc fusion protein.

[0276] Figure 27 Mice were immunized intramuscularly with 10 μg of IFN-HA1-Fc or the same molar amount of HA1 protein, and a booster immunization was administered 14 days after the initial vaccination. Serum was collected 28 days after immunization and HA1-specific antibody levels were measured by ELISA. Forty-two days after immunization, mice were intranasally infected with 1000 PFU of influenza A / PR8 virus. Starting on day three after infection, mice were observed and their weight changes were recorded. (a) Serum was collected 28 days after the initial immunization and HA1-specific antibody levels were measured by ELISA. (b) Weight changes of mice after infection.

[0277] Figure 28. SDS-PAGE electrophoresis identification of IFNa-Pan-VZV-gE-Fc, IFNa-Pan-EBV-gp350-Fc, and IFNa-Pan-HSV-2-gD-Fc proteins.

[0278] Figure 29 Compared to free EBV gp350, IFNα-Pan-EBV gp350-Fc induces a stronger humoral immune response. C57BL / 6 mice (n=5 / group) were immunized intramuscularly with 10 μg of IFNα-Pan-EBV gp350-Fc or the same molar amount of EBV gp350 protein. A booster vaccination at the same dose was administered 14 days after the primary immunization. Serum was collected 14 and 28 days after the primary immunization, and EBV gp350-specific antibody levels were measured by ELISA.

[0279] Figure 30 IFNa-Pan-HSV-2 gD-Fc elicits a stronger humoral immune response than free HSV-2 gD protein. C57BL / 6 mice (n=5) were inoculated intramuscularly with 10 μg of IFNa-Pan-HSV-2 gD-Fc or the same molar amount of HSV-2 gD protein on days 0 and 14, respectively. Serum was collected 14 and 28 days after the initial inoculation, and serum levels of HSV-2 gD-specific antibodies were measured by ELISA.

[0280] Figure 31IFNa-Pan-VZV gE-Fc elicited a stronger humoral immune response than free VZV-2 gE protein and induced a balanced Th1 / Th2 T cell immune response.

[0281] C57BL / 6 mice (n=6 / group) were immunized intramuscularly with 10 μg of IFNα-Pan-VZV gE-Fc or the same molar amount of VZV E protein. A booster immunization was administered at the same dose 14 days after the primary immunization. Serum was collected 14 and 28 days after the primary immunization, and EBV gp350-specific IgG levels were measured by ELISA (a). VZV gG-specific IgG1 (b) and IgG2c (c) subtypes were measured in serum 28 days after the primary immunization. DETAILED DESCRIPTION

[0282] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be described in detail below with reference to the following embodiments and accompanying drawings. The embodiments described herein are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The examples described herein are only a part of the present invention, not all embodiments. The scope of the present invention is defined by the specific claims appended hereto.

[0283] Example 1. Design of vaccine platform

[0284] The interferon-target antigen-immunoglobulin Fc (or antibody) structural unit vaccine platform is composed of three structural units: the first structural unit is the interferon portion, 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 via the linker sequence 2. Its representative form is as follows:

[0285] 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.

[0286] 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.

[0287] 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.

[0288] 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:

[0289] 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.

[0290] 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.

[0291] 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.

[0292] Example 2. Construction, purification and production of vaccine platform

[0293] We describe the expression and production of this vaccine platform using the hepatitis B virus Pres1 and coronavirus SARS-CoV-2 RBD protein homodimer forms as examples.

[0294] 1. Vector construction, host cell transfection and induced expression

[0295] 1.1. Using PEE12.4 as a vector, the vaccine structural unit was constructed into the vector through molecular cloning to obtain a plasmid that can express the fusion protein. The plasmid was then transiently transfected into 293F cells, the culture supernatant was collected, and the target protein was finally purified using a Protein A affinity chromatography column.

[0296] Vector construction (taking HBV preS1 antigen as an example)

[0297] (1) PEE12.4-HindIII-Signal Peptide 1-Interferon-BsiwI-Pres1-BstbI-hIgG1-EcoRI

[0298] (2) PEE12.4-HindIII-Signal Peptide 1-Interferon-BsiwI-RBD (SARS-CoV-2)-BstbI-hIgG1-EcoRI

[0299] (3) PEE12.4-HindIII-Signal Peptide 1-Interferon-Bsiwi-PADER-Pres1-hIgG1-EcoRI

[0300] (4) PEE12.4-HindIII-Signal Peptide 1-Interferon-Bsiwi-PADER-RBD(SARS-CoV-2)-hIgG1-EcoRI

[0301] The connecting sequence between each fusion protein fragment is

[0302] (1) There is a linker fragment 1 between interferon and Pres1

[0303] (2) There is a connecting segment 1 between interferon and RBD (SARS-CoV-2)

[0304] (3) The linker sequence between interferon and PADER is linker fragment 1, and the linker fragment between PADER and Pres1 is linker fragment 2

[0305] (4) The linker sequence between interferon and PADER is linker segment 1, and the linker sequence between PADER and RBD (SARS-CoV-2) is linker segment 2

[0306] 1.2. Transient transfection to rapidly express target protein:

[0307] (1) Cell recovery: Freestyle 293F cells were cultured at 3×10 7 Cryopreserved in CD OptiCHO™ media (containing 10% DMSO) at a concentration of 10 cells / ml. After removal from liquid nitrogen, rapidly thaw in a 37°C water bath, add to a 15ml centrifuge tube containing 10ml of OptiCHO™ media, and centrifuge at 1,000 rpm for 5 minutes. Discard the supernatant and resuspend the cell pellet in 30ml of OptiCHO™ media at 37°C, 8% CO2, and 135 rpm. After 4 days, expand the cells. Do not exceed a concentration of 3×10 cells / ml during expansion. 6 cells / ml.

[0308] (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.

[0309] (3) Two days later, count the transfected cell suspension and estimate the cell density to be 2.5-3.5×10 6 cells / ml, and then the cell suspension was centrifuged at 1,000 rpm for 5 min, and the supernatant was discarded.

[0310] (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.

[0311] (5) Resuspend the 293F cells in 200 ml of Freestyle 293 media.

[0312] (6) Dilute 600 μg of plasmid with 5 ml of Freestyle 293 media and sterilize by filtration using a 0.22 μM filter.

[0313] (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.

[0314] (8) Add the plasmid / PEI mixture to the cell suspension and culture in a 37°C, 8% CO2, 85rpm incubator. At the same time, add 50ug / L LONG™ R3IGF-1 growth factor.

[0315] (9) After 4 hours, add 200 ml of EX-CELL™ 293 media and 2 mM Glutamine, adjust the speed to 135 rpm and continue culturing.

[0316] (10) After 24 hours, add 3.8 mM VPA, a cell proliferation inhibitor, and 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.

[0317] 1.3. Collection, purification and electrophoresis verification of fusion proteins

[0318] 2. Purify the target protein using Protein A:

[0319] (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 NaN3 at a final concentration of 0.05% to prevent bacterial contamination during the purification process.

[0320] (2) Assemble the chromatography column: Take an appropriate amount of Protein A Agarose (calculated to purify 20 mg of human Fc fusion protein per 1 ml of Protein A), mix well, and add it to the chromatography column. Let it stand at room temperature for about 10 minutes. After the Protein A and 20% ethanol solution are separated, open the outlet at the bottom and let the ethanol solution slowly flow out by gravity.

[0321] (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.

[0322] (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.

[0323] (5) Rinse the column with more than 10 column volumes of Binding buffer to remove impurities and rinse until no protein is detected in the effluent.

[0324] (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. Observe the elution peak using a protein indicator solution (Bio-Rad protein assay). Mix the collected 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).

[0325] (7) Use a Zeba desalting spin column or a concentrating spin column to replace the target protein solution into the required buffer (note that the pH of the buffer should be adjusted to avoid the isoelectric point of the protein). Using BSA as a standard, determine the protein concentration by SDS-PAGE electrophoresis and NanoDrop2000.

[0326] (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 immerse the gel medium and store at 4°C.

[0327] 3. The protein SDS-PAGE electrophoresis identification diagram is as follows Figure 7 shown.

[0328] Example 3. IFNα-Pres1-Fc and Pres1-Fc can induce stronger immune responses in mice compared to the simple Pres1 antigen.

[0329] Materials: Male C57BL / 6 mice (5-8 weeks old) 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 plates were purchased from Corning Costar; ELISA developer was purchased from eBioscience; and the microplate reader SPECTRA max PLUS 384 was purchased from Molecular Biology, Inc. Aluminum adjuvant was purchased from SIGMA.

[0330] method:

[0331] (1) Immunization of mice with Pres1 fusion protein: 80 pmol IFN-Pres1-Fc or 80 pmol Pres1-Fc and Pres1 protein were mixed with aluminum adjuvant and immunized subcutaneously. Sera were collected from mice by orbital bleeding at designated time points for antibody detection.

[0332] (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 was administered. After 6 weeks, mice with persistent and stable HBV antigen expression were selected for the experiment. Selected mice (4 per group) were intravenously injected with 200 μl of serum from mice immunized with IFNα-Pres1-Fc. Twelve hours later, serum was collected and analyzed by ELISA for changes in Pres1 antigen expression before and after antiserum injection.

[0333] (3) ELISA detection of anti-Pres1 specific antibodies in serum. 50 μl of Pres1 (2 μg / ml) coating solution was added to an ELISA plate (Corning 9018) per well and coated overnight at 4°C. Wash once with PBS and add 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 per well to the blocked ELISA plate and incubate at 37°C for 1 hour. Wash five times with PBST, 260 μl each time, and add 50 μl of enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted in PBS) 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 TMB substrate, incubate at room temperature in the dark, and wait for the substrate to develop color; add 50 μl of stop solution (2N H2SO4) to each well to stop color development, read the plate with a microplate reader, OD450-630.

[0334] 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 As shown in (a). The antibodies induced by IFNα-Pres1-Fc can produce a wide range of neutralizing effects on different HBV genotypes, such as Figure 8 (b)

[0335] Example 4. IFNα-Pres1-Fc can be used as a preventive vaccine against hepatitis B

[0336] Materials: C57BL / 6 (6-8) week old male mice were purchased from Beijing Weitonglihua Biotechnology Co., Ltd., and the HBsAg detection kit was purchased from Shanghai Kehua Bioengineering 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.

[0337] method:

[0338] (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 serum was collected and mice were infected with 1x10 11vgAAV-HBV 1.3 virus was injected into the mice. Serum was collected weekly for four consecutive weeks to measure anti-Pres1 antibodies, HBsAg, and Pres1 antigen. HBV-DNA levels in the peripheral blood of the mice were measured at week three.

[0339] (2) ELISA detection of Pres1-specific antigen in serum. Antigen coating: 50 μl of the Pres1 antibody XY007 (4 μg / ml) coating solution was added to an ELISA plate (Corning 9018) per well and coated overnight at 4°C. Wash once with PBS and add 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 per well to the blocked ELISA plate, set up two replicates for each dilution, and incubate at 37°C for 1 hour. Wash five 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 TMB substrate, incubate at room temperature in the dark, and wait for the substrate to develop color; add 50 μl of stop solution (2N H2SO4) to each well to stop color development, read the plate with a microplate reader, OD450-630.

[0340] 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. Figure 9 (a). Compared with the group without protein immunization, IFN-Pres1-Fc vaccine immunization can significantly prevent HBV infection. The anti-preS1 antibodies produced after immunization can quickly and completely eliminate preS1 antigens in the serum. Figure 9 (b) In the IFN-Pres1-Fc immunization group, most of the mice infected with the virus showed peripheral HBsAg negative. Figure 9 (c, d). The above experimental results show that IFN-Pres1-Fc as a vaccine can effectively prevent HBV infection. Figure 9 shown.

[0341] Example 5. IFNα-Pres1-Fc as a therapeutic vaccine for chronic influenza B infection

[0342] 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 those in Example 4.

[0343] method:

[0344] (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 and used as HBV carrier mice for experiments.

[0345] (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 14 days after immunization and weekly thereafter. The levels of anti-Pres1 antibodies, HBsAg, and Pres1 antigen in the mouse serum were measured by ELISA. The HBV-DNA content in the mouse peripheral blood was measured after the final blood draw.

[0346] Results: We detected the changes in serum preS1 antigen, serum PreS1 antibodies 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. Figure 10 (a) and the preS1 antigen in the serum can be completely cleared, as shown in Figure 10 (b) As shown, at the same time, the HBsAg level in the serum also decreased to a certain extent as shown in Figure 10 (c), while the untreated control group and the Pres1 vaccine alone immunization group had no therapeutic effect. Figure 10 shown.

[0347] Example 6. T cell helper epitopes enhance the antibody response to IFNα-Pres1-Fc vaccine

[0348] Materials: Same as Example 3

[0349] method:

[0350] (1) Immunize mice with Pres1 fusion protein. 80 pmol of IFN-Pan-Pres1-Fc containing the Pan epitope or 80 pmol of IFN-Pan-Pres1-Fc, Pres1-Fc, or Pres1 protein were subcutaneously immunized. Serum was collected from the mice by orbital bleeding at the designated time points for antibody detection.

[0351] (2) Detection of anti-Pres1 specific antibodies in serum by ELISA, as in Example 3.

[0352] Results: Compared with fusion protein vaccines such as IFN-preS1-Fc, IFN-Pan-preS1-Fc significantly enhanced the immunogenicity of the antigen molecule and induced the production of broad-spectrum neutralizing antibodies. C57 / BL6 mice (n=8 / group) were subcutaneously immunized with aluminum-adjuvant-free HBV Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins. Serum Pres1-specific antibody levels were measured using ELISA at designated times.

[0353] Example 7. IFNα-Pan-Pres1-Fc as a therapeutic vaccine for chronic influenza B infection

[0354] 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 those in Example 4.

[0355] method:

[0356] (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 and used as HBV carrier mice for experiments.

[0357] (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 14 days after immunization and weekly thereafter. The levels of anti-Pres1 antibodies, HBsAG, and Pres1 antigen in the mouse serum were measured by ELISA. The HBV-DNA content in the mouse peripheral blood was measured after the final blood draw.

[0358] Results: We detected the changes in serum levels of preS1 antigen, Pres1 antibodies, 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 Figure 12 (a) and the preS1 antigen in the serum can be completely cleared. Figure 12(b) As shown, the HBsAg level in the serum also decreased to a certain extent 12 (c), while the untreated control group and the group immunized with Pres1 vaccine alone had no therapeutic effect. In addition, the HBV DNA level also decreased significantly in the IFNα-Pan-Pres1-Fc immunization group. Figure 12 (d) shown.

[0359] Example 8. IFNα-Pan-Pres1-Fc combined with HBsAg commercial vaccine breaks the immune tolerance caused by HBsAg and induces HBsAg-HBsAb seroconversion.

[0360] 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.

[0361] method:

[0362] (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 and used as HBV carrier mice for experiments.

[0363] (2) The HBV carrier mice screened were immunized with 80 pmol IFNα-pan-Pres1-Fc and 2 μg of commercial HBsAg vaccine twice, 14 days apart. Mouse serum was collected 14 days after the first immunization and weekly thereafter, and serum levels of anti-Pres1, Pres1, anti-HBsAg, and HBsAg were measured. The level of HBV-DNA in the serum was measured at the final collection of serum.

[0364] Results: We found that combining IFNα-Pan-Pres1-Fc with commercial HBsAg as a treatment strategy for chronic hepatitis B can ultimately break HBsAg tolerance. The immune response generated in HBV-tolerant mice can completely eliminate preS1 antigen in the serum. Figure 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. Figure 13 (b) and Figure 13 (d), which is clinically considered a key indicator of HBV cure. 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. Figure 13 (e) Based on the above results, we invented a vaccine strategy for the treatment of chronic hepatitis B by combining IFNα-Pan-Pres1-Fc with commercial HBsAg vaccine

[0365] Example 9. IFNα-RBD(SARS-CoV2)-Fc can induce a stronger antibody response than free SARS-CoV2 RBD protein

[0366] Materials: Balb / c male and female mice (6-8 weeks) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and SARS-CoV-2 RBD protein was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd. 293-hACE2 cells were provided by Professor Zhang Zheng (Shenzhen Third People's Hospital). Luciferase Reporter Detection Kit Purchased from Promega.

[0367] Other experimental materials are the same as those in Example 3.

[0368] method:

[0369] (1) Immunization of mice with IFNα-RBD (SARS-CoV-2)-Fc fusion protein: 10 μg of IFNα-RBD-Fc, RBD-Fc, or 10 μg of RBD protein was mixed with aluminum adjuvant and immunized subcutaneously. Serum was collected from the mice by orbital bleeding 28 days after immunization for detection of SARS-CoV-2-specific antibodies.

[0370] (2) Detection of serum SARS-CoV2 RBD antibodies. Antigen coating: Add 100 μl of RBD (1.5 μg / ml) coating solution to the ELISA plate (Corning 9018) per well and coat overnight at 4°C. Wash once with PBS, 260 μl per well. Block with 100 μl of 5% blocking solution (5% FBS) at 37°C for two hours. Dilute the serum sample with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000…), add 100 μl per well to the blocked ELISA plate and incubate at 37°C for 1 hour. Wash 5 times with PBST, 260 μl each time, add 100 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 the plate five times with PBST (260 μl each time), add 100 μl / well of the substrate TMB, and incubate at room temperature in the dark for 15 minutes to allow the substrate to develop color. Add 50 μl of stop solution (2N H₂SO₄) to each well to stop color development. Read the plate on a microplate reader, OD 450-630. To calculate the titer, select the highest dilution factor that yields a positive result. Multiply the OD value corresponding to that dilution factor by the cutoff value (0.1) and the dilution factor (X). This yields the antibody titer corresponding to the serum.

[0371] (3) In vitro neutralization experiment of SARS-CoV-2 S protein pseudovirus. Antiserum was diluted 1:3 and added to a 96-well plate. 50 μl of pseudovirus particles with luciferase spike protein were added to the wells. The virus-antibody mixture was incubated at 37°C for 1 hour. 10^4 293-hACE2 cells were added to each well of the 96-well plate. The 96-well plate was placed in a 37°C cell incubator and luciferase activity was detected after 48 hours.

[0372] Results: The immunogenicity of free SARS-CoV-2 RBD is weak. When IFNα and Fc parts are added to the SARS-CoV-2 RBD polypeptide protein region to form IFNα-RBD-Fc fusion protein, its immunogenicity is greatly improved. Figure 14 As shown. And the antibodies induced by IFNα-RBD-Fc can block the infection of SARS-CoV-2 S protein pseudovirus to cells in vitro. Figure 15 shown.

[0373] Example 10. Detection of RBD-specific antibodies in antiserum produced by immunization with FNα-Pan-RBD (original strain)-Fc and IFNα-RBD (SARS-CoV-2 South African mutant strain)-Fc.

[0374] Materials: Balb / c male and female mice (6-8 weeks) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and the RBD protein of the original SARS-CoV-2 strain was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd. The RBD protein of the South African mutant SARS-CoV-2 strain was purchased from Beijing Sino Biological Technology Co., Ltd.

[0375] Other experimental materials are the same as those in Example 3.

[0376] method:

[0377] (1) The construction and expression methods of IFNα-Pan-RBD (original strain)-Fc and IFNα-RBD (SARS-CoV-2 South African mutant strain)-Fc proteins were the same as those in Example 2.

[0378] (2) Immunization of mice with IFNα-Pan-RBD (original strain)-Fc and IFNα-Pan-RBD (SARS-CoV-2 South Africa mutant)-Fc fusion proteins. 10 μg of IFNα-Pan-RBD (original strain)-Fc or IFNα-Pan-RBD (SARS-CoV-2 South Africa mutant)-Fc protein was mixed with aluminum adjuvant and subcutaneously immunized into mice. On day 14 of immunization, serum from mice was collected by orbital bleeding for detection of SARS-CoV-2-specific antibodies.

[0379] (3) Analysis of antibody response by ELISA method is the same as in Example 9.

[0380] Results: SDS-PAGE results showed the correct band size of IFNα-Pan-RBD (SARS-CoV-2 original strain)-Fc, indicating that the mutant new coronavirus IFNα-RBD (SARS-CoV-2 original strain)-Fc vaccine protein was successfully constructed, expressed, and purified ( Figure 16 a) SDS-PAGE results showed the correct band size of IFNα-Pan-RBD (SARS-CoV-2 South Africa mutant)-Fc, indicating that the mutant novel coronavirus IFNα-RBD (SARS-CoV-2 South Africa mutant)-Fc vaccine protein was successfully constructed, expressed, and purified ( Figure 16 b). ELISA results showed that the antibodies induced by mice immunized with IFNα-Panan-RBD (original strain)-Fc and mice immunized with IFNα-Pan-RBD (SARS-CoV-2 South African mutant)-Fc could bind to the RBD protein of the original strain of the new coronavirus, and there was no significant difference in the binding ability of the antibodies induced by the two to the RBD protein of the original strain ( Figure 16c). At the same time, the ELISA results for the South African mutant RBD also showed that the antibodies induced by mice immunized with IFNα-Panan-RBD (original strain)-Fc and mice immunized with IFNα-Pan-RBD (SARS-CoV-2 South African mutant)-Fc can bind to the South African mutant RBD with comparable binding abilities ( Figure 16 d).

[0381] Example 11.

[0382] Materials: C57BL / 6 female mice (6-8 weeks) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. The SARS-CoV-2 RBD protein used in ELISA was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd.; the mouse IFNα-RBD-Fc, mouse IFNα-Pan-RBD-Fc, human IFNα-RBD-Fc, and human IFNα-Pan-RBD-Fc proteins used for immunization were all produced in our laboratory. Other experimental materials were the same as those in Example 3.

[0383] method:

[0384] (1) The fusion protein design, plasmid construction and protein purification methods are the same as those in Examples 1 and 2.

[0385] (2) Immunization of mice with vaccine proteins. 10 μg of mouse IFNα-RBD-Fc, mouse IFNα-Pan-RBD-Fc, or 10 μg of human IFNα-RBD-Fc, human IFNα-Pan-RBD-Fc vaccine protein was mixed with 20 μg of aluminum adjuvant overnight and then inoculated into mice via intramuscular immunization. A booster immunization was performed 14 days after the initial vaccination. Mouse sera were collected on days 7, 14, and 28 after immunization, and the levels of RBD-specific antibodies in the mouse sera were detected by ELISA.

[0386] (3) Detection of serum SARS-CoV2 RBD antibodies. Antigen coating: Add 100 μl of RBD (1.5 μg / ml) coating solution to the ELISA plate (Corning 9018) per well and coat overnight at 4°C. Wash once with PBS, 260 μl per well. Block with 100 μl of 5% blocking solution (5% FBS) at 37°C for two hours. Dilute the serum sample with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000…), add 100 μl per well to the blocked ELISA plate and incubate at 37°C for 1 hour. Wash 5 times with PBST, 260 μl each time, add 100 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 the plate five times with PBST (260 μl each time), add 100 μl / well of the substrate TMB, and incubate at room temperature in the dark for 15 minutes to allow the substrate to develop color. Add 50 μl of stop solution (2N H₂SO₄) to each well to stop color development. Read the plate on a microplate reader, OD 450-630. To calculate the titer, select the highest dilution factor that yields a positive result. Multiply the OD value corresponding to that dilution factor by the cutoff value (0.1) and the dilution factor (X). This yields the antibody titer corresponding to the serum.

[0387] result:

[0388] like Figure 17 As shown in the figure, after the protein was expressed and purified, SDS-PAGE results showed that the protein size was consistent with the expectation and exhibited a single band at the target position.

[0389] like Figure 18 As shown, the addition of the Pan (Pan DR-binding epitope) CD4 T cell helper epitope can enhance the immunity of Mouse IFNα-RBD-Fc and Human IFNα-RBD-Fc. Experimental results show that whether on day 7, day 14, or day 28 after vaccine protein immunization, the addition of the Pan epitope can induce higher production of RBD-specific antibodies in Mouse IFNα-Pan-RBD-Fc compared to Mouse IFNα-RBD-Fc, and in Human IFNα-Pan-RBD-Fc compared to Human IFNα-RBD-Fc.

[0390] Example 12

[0391] Materials: C57BL / 6 female mice (6-8 weeks) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd.; SARS-CoV-2 RBD protein used in ELISA was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd. Human IFNα-RBD-Fc and human IFNα-Pan-RBD-Fc proteins used for immunization were produced in our laboratory. Other experimental materials were the same as in Example 3.

[0392] method:

[0393] (1) Immunization of mice with Human IFNα-RBD-Fc or Human IFNα-Pan-RBD-Fc proteins. 10 μg of Human IFNα-RBD-Fc or Human IFNα-Pan-RBD-Fc protein was mixed with aluminum adjuvant overnight and used as vaccine samples containing aluminum adjuvant. Another group of 10 μg of Human IFNα-RBD-Fc or Human IFNα-Pan-RBD-Fc protein was diluted with PBS and used as vaccine samples without adjuvant. Mice were inoculated with 10 μg of Human IFNα-RBD-Fc or Human IFNα-Pan-RBD-Fc protein by intramuscular immunization in the presence or absence of aluminum adjuvant, and a booster immunization was performed 14 days after vaccination. Mouse sera were collected on days 7, 14, and 28 after immunization, and the levels of RBD-specific antibodies in the mouse sera were detected by ELISA.

[0394] (2) Detection of serum SARS-CoV2 RBD antibodies. Antigen coating: Add 100 μl of RBD (1.5 μg / ml) coating solution to the ELISA plate (Corning 9018) per well and coat overnight at 4°C. Wash once with PBS, 260 μl per well. Block with 100 μl of 5% blocking solution (5% FBS) at 37°C for two hours. Dilute the serum sample with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000…), add 100 μl per well to the blocked ELISA plate and incubate at 37°C for 1 hour. Wash 5 times with PBST, 260 μl each time, add 100 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 the plate five times with PBST (260 μl each time), add 100 μl / well of the substrate TMB, and incubate at room temperature in the dark for 15 minutes to allow the substrate to develop color. Add 50 μl of stop solution (2N H₂SO₄) to each well to stop color development. Read the plate on a microplate reader, OD 450-630. To calculate the titer, select the highest dilution factor that yields a positive result. Multiply the OD value corresponding to that dilution factor by the cutoff value (0.1) and the dilution factor (X). This yields the antibody titer corresponding to the serum.

[0395] result:

[0396] like Figure 19 As shown in the results, the use of aluminum adjuvants can enhance the immunogenicity of Human IFNα-RBD-Fc and Human IFNα-Pan-RBD-Fc proteins. Although the unadjuvanted Human IFNα-RBD-Fc and Human IFNα-Pan-RBD-Fc vaccines can generate high-titer antibody responses, the aluminum adjuvant-assisted Human IFNα-RBD-Fc and Human IFNα-Pan-RBD-Fc proteins can further enhance the RBD-specific antibody response level on days 7, 14, and 28 after vaccination compared with the unadjuvanted group.

[0397] Example 13.

[0398] Material:

[0399] Experimental animals were purchased from Beijing Weitonglihua Laboratory Animal Co., Ltd., and the animals used were 6- to 8-week-old C57BL / 6 mice; animal qualification certificate number: No. 110011200106828974; RBD protein used for immunization was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd.; RBD-Fc, IFNα-RBD-Fc, and IFN-pan-RBD-Fc proteins were produced in our laboratory; all adjuvants were purchased from SERVA, Germany; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Beijing Kangwei Biotechnology Co., Ltd.; 96-well ELISA plates were purchased from Corning Costar; ELISA developer was purchased from eBioscience; the microplate reader SPECTRA max PLUS 384 was purchased from Molecular Biology, USA; and a tissue homogenizer was purchased from Beijing Haonuosi Technology Co., Ltd.

[0400] method:

[0401] Six- to eight-week-old mice were divided into five groups of ten and immunized intranasally with 10 μg of IFNα-pan-RBD-Fc or equivalent molar amounts of RBD, RBD-Fc, or IFNα-RBD-Fc protein (10 μL per mouse). Mice were immunized twice, on days 0 and 14. Serum was collected on days 7, 14, 21, 28, 35, and 42 after immunization, and SARS-CoV-2 RBD-specific antibodies were assayed by ELISA. Serum collected on day 28 was used for in vitro neutralization of SARS-CoV-2 pseudoviruses.

[0402] result:

[0403] like Figure 20 As shown, two intranasal immunizations of RBD and RBD-Fc proteins can induce a certain degree of antibody response. The serum IgG and IgA levels induced by IFNα-pan-RBD-Fc at the same time point after two intranasal immunizations were significantly higher than those in the RBD, RBD-Fc and IFN-RBD-Fc groups. The results of the pseudovirus neutralization experiment showed that compared with the RBD and RBD-Fc immunization groups, IFN-RBD-Fc can induce higher levels of neutralizing antibodies.

[0404] Example 14.

[0405] Material:

[0406] Same as Example 10

[0407] method:

[0408] Six- to eight-week-old mice were divided into four groups of five and immunized intranasally with 10 μg of IFNα-pan-RBD-Fc or equivalent molar amounts of RBD, RBD-Fc, or IFNα-RBD-Fc protein (10 μL per mouse). Mice were immunized twice, on days 0 and 14. Nasal mucosal supernatants and lung lavage fluids were collected on day 28 after immunization. Serum from each group was assayed for SARS-CoV-2 RBD-specific antibodies using an ELISA. Serum and nasal mucosal supernatants were tested for SARS-CoV-2 pseudovirus neutralization using a SARS-CoV-2 pseudovirus neutralization assay.

[0409] Obtaining nasal mucosal supernatant and bronchoalveolar lavage fluid from mice for the immunization experiment: After dormant mice were sacrificed, the nasal mucosa was obtained and disrupted using a tissue homogenizer. The homogenized fluid was centrifuged at 13,000 rpm for 10 minutes. The supernatant was obtained as the nasal mucosal supernatant (NMDS). For the mouse lungs, approximately 0.8 ml of HBSS + 100 μM EDTA was drawn into the endotracheal cannula using a 1 ml syringe. After three gentle aspirations, the fluid was aspirated and collected into a centrifuge tube. This step was repeated three times to obtain approximately 2 ml of bronchoalveolar lavage fluid. The bronchoalveolar lavage fluid was centrifuged at 500 g for 5 minutes. The supernatant was obtained as the bronchoalveolar lavage fluid (BALF). Lymphocytes present in the mouse lungs were precipitated for further analysis.

[0410] result:

[0411] like Figure 21 As shown, compared with RBD and RBD-Fc proteins, two intranasal immunizations with IFNα-pan-RBD-Fc protein elicited robust local IgG antibody responses and IgA mucosal immunity in the nasal mucosa. The intensity of the IFNα-pan-RBD-Fc protein response was stronger than that of the RBD and RBD-Fc groups. Pseudovirus neutralization experiments showed that the IFNα-pan-RBD-Fc protein immunization group induced higher titers of neutralizing antibodies in the nasal mucosa.

[0412] like Figure 22 As shown, two intranasal immunizations of C57BL / 6 mice with the IFNα-pan-RBD-Fc fusion protein also induced strong secretion of IgG and IgA antibodies in local lung tissue. Pseudovirus neutralization experiments showed that IFNα-pan-RBD-Fc induced higher titers of neutralizing antibodies than RBD and RBD-Fc.

[0413] Example 15.

[0414] Her2 belongs to the HER family of type I transmembrane growth factor receptors and is composed of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. Upon ligand binding to the extracellular domain, the HER protein dimerizes and transphosphorylates its intracellular domain. Phosphorylated tyrosine residues can bind to various intracellular signaling molecules, activating downstream signaling pathways and regulating gene transcription. The regulated genes are often involved in processes such as cell proliferation, survival, differentiation, angiogenesis, invasion, and metastasis. The extracellular region of the HER2 protein is large, comprising over 600 amino acids, and is divided into four domains: domains I, II, III, and IV. The currently approved trastuzumab binds primarily to domain IV, pertuzumab binds primarily to domain II, and the peptide vaccine E75, currently in clinical trials, targets domain III. This suggests that each domain contains important sites that may mediate anti-tumor effects. In order to study the use of this vaccine platform for tumor prevention and treatment, this patent study selected the tumor antigen Her2 as the target, constructed IFN-Her2-Fc and IFN-Pan-Her2-Fc fusion protein vaccines, and analyzed the in vivo anti-tumor activity and vaccine immune activity.

[0415] Materials and methods:

[0416] Material:

[0417] BALB / c female mice (6-8 weeks) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd.; TUBO cells were obtained from TCGA; other materials were the same as in Example 3.

[0418] method:

[0419] (1) The fusion protein design, plasmid construction and protein purification methods are shown in Examples 1 and 2.

[0420] First, expression plasmids were constructed targeting domains III and IV of the mouse Her2 extracellular domain (denoted as IFNα-3-Fc, IFNα-pan-3-Fc, IFNα-pan-4-Fc, and IFNα-4-Fc, respectively). The proteins were expressed and purified in the human 293F cell line. Protein size and purity were assessed using SDS-PAGE and Coomassie Brilliant Blue staining.

[0421] (2) Analysis of the direct antitumor activity of IFNα-3-Fc and IFNα-pan-3-Fc

[0422] TUBO is a breast cancer cell line derived from BALB-NeuT mice and is used to study the growth and treatment of Her2-positive breast cancer. The anti-tumor activity of IFNα in TUBO tumor detection protein was determined. TUBO breast cancer model mice were constructed with 5*10 5TUBO cells were subcutaneously inoculated into BALB / C mice with tumors ranging in size from 50 to 80 mm. 3 Treatment was administered once weekly for a total of three times. The dose of IFNα-3-Fc was 10 μg / mouse, and other drugs were administered in equal moles. CpG was used as an adjuvant. Tumor size was measured, and tumor growth curves were plotted.

[0423] (3) Analysis of the effect of IFNα and Pan on the immunogenicity of Her2 vaccine

[0424] BALB / C female mice aged 6-8 weeks were subcutaneously inoculated with the unadjuvanted HER2 domain V fusion protein vaccines 4-Fc, IFNα-4-Fc, and IFNα-pan-4-Fc once weekly for a total of three doses. The immunization dose was 10 μg / mouse of IFNα-4-Fc, and the other proteins were administered at equal molar amounts. Venous blood was collected 14 and 21 days after immunization, and HER2-specific IgG antibody levels were measured by ELISA.

[0425] result:

[0426] (1) If Figure 23 As shown, the Her2 fusion proteins are generally in expected size and meet experimental requirements for purity. IFNα-3-Fc (62.6 kDa), IFNα-pan-3-Fc (63.9 kDa), IFNα-pan-4-Fc (74.9 kDa), and IFNα-4-Fc (73.6 kDa) are dimers under non-deforming conditions, consistent with the auto-dimerization property of Fc fragments.

[0427] (2) If Figure 24 As shown in the results, compared with the control group, intratumoral injection of the Her2 fusion proteins IFNα-pan-3-Fc and IFNα-3-Fc significantly inhibited the growth of TUBO tumors, and the control effect was comparable to that of the IFNα-Fc group. This indicates that the IFNα activity in the protein vaccine is good and that there are no factors such as steric hindrance that affect its IFNα activity, which can be used to further explore its efficacy and mechanism in anti-tumor immunity.

[0428] (3) If Figure 25As shown, compared with the control group, 14 and 21 days after immunization with the Her2 fusion protein vaccine, 4-Fc, IFNα-4-Fc, and IFNα-pan-4-Fc all induced significant Her2-specific IgG antibody responses. Compared with 4-Fc, the antibody titers induced by IFNα-4-Fc and IFNα-pan-4-Fc showed an increasing trend. Furthermore, at 21 days after immunization, the antibody titers induced by IFNα-pan-4-Fc were significantly higher than those in the 4-Fc group. This suggests that the addition of IFNα and pan helps enhance the immunogenicity of 4-Fc, inducing a stronger antigen-specific antibody response. Therefore, IFN-Pan-HER2-Fc and IFN-Pan-HER2-Fc are potentially effective tumor vaccines against Her2-positive tumors.

[0429] Example 16.

[0430] Materials: BALB / c female mice (6-8 weeks) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd.; HA1 (A / PR8) protein used for ELISA was purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.; HA1 protein (A / PR8) used for immunization was purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., and IFNα-HA1-Fc was produced in our laboratory; the H1N1 (A / PR8) influenza virus used to infect mice was produced in our laboratory; other experimental materials were the same as those in Example 3.

[0431] method:

[0432] (1) The design, plasmid construction and protein purification of IFNα-HA1-Fc protein were the same as those in Examples 1 and 2.

[0433] (2) Immunization of mice with HA1 or IFNα-HA1-Fc proteins. 10 μg of IFNα-HA1-Fc or the same molar amount of HA1 protein was mixed with 20 μg of aluminum adjuvant overnight and then inoculated into mice by intramuscular immunization. A booster immunization was performed 14 days after the initial vaccination. Serum was collected from mice on day 28 after immunization, and the HA1-specific antibody level in the serum was detected by ELISA.

[0434] (3) Detection of serum HA1 antibodies. Antigen coating: Add 100 μl of HA1 (2 μg / ml) coating solution to each well of the Elisa plate (Corning 9018) and coat overnight at 4°C. Wash once with PBS, 260 μl per well. Block with 100 μl of 5% blocking solution (5% FBS) at 37°C for two hours. Dilute the serum sample with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000…), add 100 μl per well to the blocked Elisa plate and incubate at 37°C for 1 hour. Wash five times with PBST, 260 μl each time, add 100 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 the plate five times with PBST (260 μl each time), add 100 μl / well of the substrate TMB, and incubate at room temperature in the dark for 15 minutes to allow the substrate to develop color. Add 50 μl of stop solution (2N H₂SO₄) to each well to stop color development. Read the plate on a microplate reader, OD 450-630. To calculate the titer, select the highest dilution factor that yields a positive result. Multiply the OD value corresponding to that dilution factor by the cutoff value (0.1) and the dilution factor (X). This yields the antibody titer corresponding to the serum.

[0435] (4) 42 days after immunization, the mice were anesthetized and infected with 1000 PFU A / PR8 influenza virus through nasal drops. Starting from the third day after infection, the mice were observed and weighed every two days.

[0436] result:

[0437] like Figure 26 As shown in the figure, after the protein was expressed and purified, the size and purity of the protein were detected by SDS-PAGE. The results showed that a single band was present at the target band size position. Figure 27 As shown, IFNα-HA1-Fc can induce higher titers of HA1-specific antibodies compared with HA1 protein ( Figure 27 a), indicating that the vaccine platform can enhance the immunogenicity of HA1 protein. The weight of mice will change significantly after the virus is challenged, but the weight of mice in the IFNα-HA1-Fc immunization group will recover rapidly compared with the PBS group and the HA1 protein immunization group, indicating that IFNα-HA1-Fc vaccine immunization has good protection against influenza infection ( Figure 27 b).

[0438] Example 17

[0439] Materials and methods:

[0440] The design, plasmid construction and protein purification of IFNa-Pan-VZV-gE-Fc, IFNa-Pan-EBV-gp350-Fc and IFNa-Pan-HSV-2-gD-Fc proteins were the same as those in Examples 1 and 2.

[0441] result:

[0442] like Figure 28 As shown, after the IFNa-Pan-VZV-gE-Fc, IFNa-Pan-EBV-gp350-Fc, and IFNa-Pan-HSV-2-gD-Fc fusion proteins were expressed and purified, the size and purity of the proteins were detected by SDS-PAGE, and the results showed that the target bands were correctly located.

[0443] Example 18

[0444] IFNa-Pan-EBV gp350-Fc induces a stronger humoral immune response than free EBV-gp350 protein

[0445] Materials: C57BL / 6 female mice (6-8 weeks) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd.; EBV-gp350 protein used for ELISA was purchased from Taizhou Baiying Biotechnology Co., Ltd.; EBV-gp350 protein used for immunization was purchased from Taizhou Baiying Biotechnology Co., Ltd., and IFNα-Pan-EBV gp350-Fc was produced in our laboratory; other experimental materials were the same as those in Example 3.

[0446] method:

[0447] (1) Mice were immunized with EBV gp350 or IFNα-Pan-EBV gp350-Fc proteins. 10 μg of IFNα-Pan-EBV gp350-Fc or the same molar amount of EBV-gp350 protein was mixed with 20 μg of aluminum adjuvant overnight and then inoculated intramuscularly. A booster immunization was performed 14 days after the initial vaccination. Serum was collected on days 14 and 28 after the initial vaccination, and the EBV-gp350-specific antibody levels in the serum were detected by ELISA.

[0448] (2) Detection of serum EBV-gp350 antibodies. Antigen coating: 100 μl of EBV-gp350 (1 μg / ml) coating solution was added to an ELISA plate (Corning 9018) at each well and coated overnight at 4°C. Wash once with PBS and add 260 μl to each well. Block with 100 μl of 5% blocking solution (5% FBS) at 37°C for two hours. Dilute the serum sample with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000…), add 100 μl to each well of the blocked ELISA plate and incubate at 37°C for 1 hour. Wash the plate five times with 260 μl of PBST each time. Add 100 μl of enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP diluted 1:5000 in PBS) to each well and incubate at 37°C for 0.5 hour. Wash the plate five times with 260 μl of PBST each time. Add 100 μl of TMB substrate per well and incubate at room temperature in the dark for 15 minutes to allow the substrate to develop color. Add 50 μl of stop solution (2N H₂SO₄) to each well to stop color development. Read the plate on a microplate reader at an OD of 450-630. To calculate the titer, select the highest dilution that yields a positive result. Multiply the OD value corresponding to that dilution by the cutoff value (0.1) and the dilution factor (X). This yields the antibody titer corresponding to that serum.

[0449] Results: As Figure 29 As shown in the figure, compared with free EBV-gp350 protein, IFNα-Pan-EBV gp350-Fc protein can induce stronger specific humoral immune response in both single immunization (Prime) and two immunizations (boost), which fully demonstrates that this vaccine platform can significantly compete for the immunogenicity of EBV-gp350.

[0450] Example 19

[0451] IFNa-Pan-HSV-2 gD-Fc can induce a stronger humoral immune response than free HSV-2 gD protein.

[0452] Materials: C57BL / 6 female mice (6-8 weeks) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd.; HSV-2 gD protein used for ELISA was purchased from Taizhou Baiying Biotechnology Co., Ltd.; HSV-2 gD protein used for immunization was purchased from Taizhou Baiying Biotechnology Co., Ltd., and IFNa-Pan-HSV-2 gD-Fc was produced in our laboratory; other experimental materials were the same as those in Example 3.

[0453] method:

[0454] (1) Mice were immunized with HSV-2 gD or IFNa-Pan-HSV-2 gD-Fc proteins. 10 μg IFNa-Pan-HSV-2 gD-Fc or the same molar amount of HSV-2 gD protein was mixed with 20 μg aluminum adjuvant overnight and then inoculated intramuscularly. A booster immunization was performed 14 days after the initial vaccination. Serum was collected on days 14 and 28 after the initial vaccination, and the EBV-gp350-specific antibody level in the serum was detected by ELISA.

[0455] (2) Detection of serum HSV-2 gD antibodies. Antigen coating: Add 100 μl of HSV-2 gD (1 μg / ml) coating solution to an ELISA plate (Corning 9018) and coat overnight at 4°C. Wash once with PBS and add 260 μl to each well. Block with 100 μl of 5% blocking solution (5% FBS) at 37°C for two hours. Dilute the serum sample with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000…), add 100 μl to each well of the blocked ELISA plate and incubate at 37°C for 1 hour. Wash the plate five times with 260 μl of PBST each time. Add 100 μl of enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP diluted 1:5000 in PBS) to each well and incubate at 37°C for 0.5 hour. Wash the plate five times with 260 μl of PBST each time. Add 100 μl of TMB substrate per well and incubate at room temperature in the dark for 15 minutes to allow the substrate to develop color. Add 50 μl of stop solution (2N H₂SO₄) to each well to stop color development. Read the plate on a microplate reader at an OD of 450-630. To calculate the titer, select the highest dilution that yields a positive result. Multiply the OD value corresponding to that dilution by the cutoff value (0.1) and the dilution factor (X). This yields the antibody titer corresponding to that serum.

[0456] Results: As Figure 30 As shown in the results, compared with free HSV-2 gD protein, IFNa-Pan-HSV-2 gD-Fc protein can induce stronger specific humoral immune response in both single immunization (Prime) and two immunizations (boost), which fully demonstrates that this vaccine platform can significantly enhance the immunogenicity of HSV-2 gD.

[0457] Example 20: IFNa-Pan-VZV gE-Fc can induce a stronger humoral immune response than free VZV-2 gE protein and can induce a Th1 / Th2 balanced T cell immune response.

[0458] Materials: C57BL / 6 female mice (6-8 weeks) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd.; VZV gE protein used for ELISA was purchased from Taizhou Baiying Biotechnology Co., Ltd.; VZV-gE protein used for immunization was purchased from Taizhou Baiying Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG1 / IgG2c for antibody subtype detection was purchased from Proteintech; IFNa-Pan-VZV gE-Fc was produced in our laboratory; other experimental materials were the same as those in Example 3.

[0459] method:

[0460] (1) VZV gE and IFNa-Pan-VZV gE-Fc proteins were used to immunize mice. 10 μg IFNa-Pan-VZV gE-Fc or the same molar amount of VZV gE protein was mixed with 20 μg aluminum adjuvant overnight and then inoculated into the mice by intramuscular immunization. A booster immunization was performed 14 days after the initial vaccination. Mouse sera were collected on the 14th and 28th days after the initial immunization, and the VZV gE-specific IgG level in the mouse sera was detected by ELISA. VZV gE-specific antibody subtypes were detected by ELISA 28 days after the initial immunization.

[0461] (2) Detection of total serum VSV gE antibodies. Antigen coating: Add 100 μl of VSV gE (1 μg / ml) coating solution to an ELISA plate (Corning 9018) and coat overnight at 4°C. Wash once with PBS, then add 260 μl to each well. Block with 100 μl of 5% blocking solution (5% FBS) at 37°C for two hours. Dilute the serum sample with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000…), add 100 μl to each well of the blocked ELISA plate, and incubate at 37°C for 1 hour. Wash five times with PBST, 260 μl each time, and add 100 μl of enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted in PBS) to each well. Incubate at 37°C for 0.5 hour. Wash the plate five times with PBST, adding 260 μl of TMB substrate per well. Incubate at room temperature in the dark for 15 minutes to allow the substrate to develop color. Add 50 μl of stop solution (2N H₂SO₄) to each well to stop color development. Read the plate on a microplate reader, OD 450-630. To calculate the titer, select the highest dilution that yields a positive result. Multiply the OD value corresponding to that dilution by the cutoff value (0.1) and the dilution factor (X). This yields the antibody titer corresponding to the serum.

[0462] (3) Detection of serum VSV gE-specific antibody subtypes. Antigen coating: Add 100 μl of VSV gE (1 μg / ml) coating solution to an ELISA plate (Corning 9018) and coat overnight at 4°C. Wash once with PBS and add 260 μl to each well. Block with 100 μl of 5% blocking solution (5% FBS) at 37°C for two hours. Dilute the serum sample with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000) and add 100 μl to each well of the blocked ELISA plate and incubate at 37°C for 1 hour. Wash five times with PBST, 260 μl each time, and add 100 μl of enzyme-labeled secondary antibody (horseradish peroxidase (HRP)-labeled goat anti-mouse IgG1 / IgG2c) to each well and incubate at 37°C for 0.5 hour. Wash the plate five times with PBST, using a 260µl volume per well. Add 100µl of TMB substrate per well and incubate at room temperature in the dark for 15 minutes to allow the substrate to develop color. Add 50µl of stop solution (2N H₂SO₄) to each well to stop color development. Read the plate on a microplate reader, using an OD range of 450-630. To calculate the titer, select the highest dilution factor that yields a positive result. Multiply the OD value corresponding to that dilution factor by the cutoff value (0.1) and the dilution factor to obtain the antibody titer corresponding to that serum.

[0463] Results: Compared with free VZV-gE protein, IFNa-Pan-VZV-gE-Fc protein induced stronger specific humoral immune response in both single immunization (Prime) and double immunization (Boost). Figure 31 a), IFNa-Pan-VZV-gE-Fc immunization can not only induce the production of Th2-biased IgG1, but also induce the production of high levels of Th1-biased IgG2c ( Figure 31 bc), while VZV-gE immunization only induced the production of Th2-biased IgG1 and could not effectively produce Th1-biased IgG2c. The above results fully demonstrate that IFNa-Pan-VZV gE-Fc can not only produce a stronger humoral immune response than free VZV-gE protein, but also induce a balanced Th1 / Th2 immune response.

[0464] References:

[0465] [1]WHO W. Global hepatitis report 2017 [J]. Geneva: World HealthOrganization, 2017,

[0466] [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.

[0467] [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.

[0468] [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.

[0469] [5]KRAMMER F, SMITH G J D, FOUCHIER R A M, et al. Influenza [J].Nature reviews Disease primers, 2018, 4(1): 3.

[0470] [6]HANNOUN C. The evolving history of influenza viruses and influenzavaccines [J]. Expert review of vaccines, 2013, 12(9): 1085-94.

[0471] [7]HOUSER K, SUBBARAO K. Influenza vaccines: challenges and solutions[J]. Cell host & microbe, 2015, 17(3): 295-300.

[0472] [8]HU B, GUO H, ZHOU P, et al. Characteristics of SARS-CoV-2 andCOVID-19 [J]. Nature Reviews Microbiology, 2020, 1-14.

[0473] [9]LURIE N, SAVILLE M, HATCHETT R, et al. Developing Covid-19vaccines at pandemic speed [J]. New England Journal of Medicine, 2020, 382(21): 1969-73.

[0474]

[10] LI Q, WU J, NIE J, et al. The impact of mutations in SARS-CoV-2spike on viral infectivity and antigenicity [J]. Cell, 2020, 182(5): 1284-94.e9.

[0475]

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

[0476]

[12] 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.

[0477]

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

[0478]

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

[0479]

[15] 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.

[0480]

[16] 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.

[0481]

[17] 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.

[0482]

[18] 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.

[0483]

[19] 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.

[0484]

[20] 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.

[0485]

[21] 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.

[0486]

[22] 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.

[0487]

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

[0488]

[24] 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 comprising interferon-target antigen-immunoglobulin Fc region (or antibody Ab) as structural units, The interferon is the first structural unit, which can be type I interferon, type II interferon and / or 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 interferon can be of human or mouse origin. Preferably, the interferon is type I interferon, such as IFN-α, such as mouse IFN-α4, human IFN-α2, a mutant of human IFN-α2 (binding to human and mouse IFN receptors), such as those shown in the amino acid sequences of SEQ ID NO.1, SEQ ID NO.21, and SEQ ID NO.

22. Wherein the target antigen is the third structural unit, and the target antigen can be, for example, a tumor antigen, a pathogen antigen, such as a virus or bacterial antigen, wherein the target antigen can be, for example, a mutated target antigen different from the wild type, including, for example, natural point mutations / deletion mutations / addition mutations / truncations of the wild type antigen, artificial point mutations / deletion mutations / addition mutations / truncations, any combination of natural or artificial mutations, and subtypes produced after mutation, wherein the virus can be, for example, SARS-COV-2, or wherein the target antigen can be, for example, the full length or S1 region of the SARS-COV-2 virus S protein, for example, the target antigen can be the antigen shown in SEQ ID NO.76 or SEQ ID NO.77, The immunoglobulin Fc region (or antibody) is the second structural unit, which can be the constant region amino acid sequence of IgG1, IgG2, IgG3, IgG4 and / or IgM, such as the Fc region of IgG1, and the Fc region shown in SEQ ID NO.2, SEQ ID NO.23, and SEQ ID NO.24 of the amino acid sequences of IgG1-Fc-hole and IgG1-Fc-knob for forming heterodimers. The antibody (including, for example, antibody heavy and light chains, or single-chain antibodies, referred to as Ab) as the second structural unit can be a DC-targeting activation antibody, including anti-PD-L1, anti-DEC205, anti-CD80 / 86 and other antibodies. Optionally, the vaccine may be a targeted vaccine, and optionally, the fusion protein may further comprise one or more Th cell helper epitopes and / or linker fragments.

2. The vaccine according to claim 1, wherein the target antigen is a viral antigen, and the virus can be, for example, HBV, HPV, VZV, EBV, HSV-2, HIV, influenza virus, coronavirus, such as SARS-CoV, SARS-COV-2, MERS-CoV, for example, the antigen can be an HBV antigen, such as HBV Pres1 antigen, HBsAg antigen or peptide, such as ad subtype or ay subtype HBVPres1 antigen, such as the ad subtype HBV Pres1 antigen shown in the amino acid sequence of SEQ ID NO.6, such as the ay subtype HBV Pres1 antigen shown in the amino acid sequence of SEQ ID NO.26; for example, HBV HBsAg antigen (including various subtypes and peptides), such as the adr subtype HBV HBsAg antigen shown in the amino acid sequence of SEQ ID NO.7, such as the adw subtype HBV HBsAg antigen shown in the amino acid sequence of SEQ ID NO.27, such as the adw subtype HBV HBsAg antigen shown in the amino acid sequence of SEQ ID NO. NO.28 amino acid sequence shown in the ayw subtype HBVHBsAg antigen; for example, the antigen can be, for example, a SARS-COV-2 antigen, such as a SARS-COV2 RBD antigen, such as the SARS-COV2 RBD antigen shown in the SEQ ID NO.8 amino acid sequence; for example, an influenza virus antigen, such as an influenza virus HA antigen, such as the influenza virus HA antigen shown in the SEQ ID NO.9 amino acid sequence; for example, an HPV antigen, such as the HPV E7 antigen shown in the SEQ ID NO.10 amino acid sequence; for example, a gE antigen, such as the herpes zoster virus (VZV) gE antigen shown in the SEQ ID NO.91 amino acid sequence; for example, EBV-gp350, such as the Epstein-Barr virus (EBV) gp350 protein shown in the SEQ ID NO.92 amino acid sequence; for example, a gD antigen, such as the herpes simplex virus 2 (HSV-2) gD antigen shown in the SEQ ID NO.93 amino acid sequence; the antigen can be, for example, EBV EBNA1 / LMP2, VZV-IE62, HSV-2 ICP0, HIV gp120 antigen, The target antigen may be a mutant viral antigen, such as a mutant of any of the aforementioned viral antigens, such as a mutant of SARS-COV-2, including, for example, natural point mutations / deletion mutations / addition mutations / truncations of SARS-COV-2 proteins (such as one or more of S protein, N protein, M protein, and E protein), artificial point mutations / deletion mutations / addition mutations / truncations, any combination of natural or artificial mutations, and subtypes produced by mutations. For example, the mutant viral antigen may be the full length S protein (SEQ ID NO. 76), S1 region (SEQ ID NO. 77), RBD region (SEQ ID NO. 78) of wild-type SARS-COV-2. NO.78), for example, the mutated viral antigen may include one or more of the following mutations of the S protein of SARS-COV-2: NTD region 69-70 deletion, Y144 deletion, 242-244 deletion, L18F, D80A, D215, R246I mutations, RBD region K417, E484, N501Y, L452R mutations, D614G, H655Y mutations, for example, the mutated viral antigen may include mutations present in the British B.1.1.7 (501Y.1) mutant strain, the South African B.1.351 (501Y.2) mutant strain and the Brazilian P1 (501Y.3) mutant strain, the California B.1.429 mutant strain, for example, the mutated viral antigen may include a mutant of any one of SEQ ID NO.79, SEQ ID NO.80, SEQ ID NO.81, and SEQ ID NO.82, for example, the mutated viral antigen may be a mutant comprising SEQ A mutant of any one of SEQ ID NO.79, SEQ ID NO.80, SEQ ID NO.81, and SEQ ID NO.82, The viral antigen may be fused with an auxiliary polypeptide epitope that enhances B cell and T cell responses, which may be located at the N-terminus or C-terminus of the antigen epitope, such as the Pan HLA DR-binding epitope (PADER), as shown in its amino acid sequence in SEQ ID NO. 3; The connecting segments of the structural units are flexible polypeptide sequences, which can be connecting segments 1 and 2, such as those shown in the amino acid sequences of SEQ ID NO. 4 and SEQ ID NO.

25. Each polypeptide sequence composed of the structural unit may contain a corresponding signal peptide at the N-terminus that can promote protein secretion, such as the amino acid sequence shown in SEQ ID NO.

5. The vaccine can be produced by a eukaryotic expression system, for example, by a eukaryotic expression system 293F or CHO cells.

3. The vaccine according to claim 1 or 2, wherein the target antigen is a tumor antigen, such as a protein molecule highly expressed in tumor cells, for example, the antigen can be human epidermal growth factor receptor 2 (HER2 / neu) and epidermal growth factor (EGFR); for example, the protein molecule Her2 highly expressed in tumor cells and its various functional regions and truncations, such as the antigens shown in SEQ ID NOs. 85, 86, 97, 88, 89, 90 and their mutants.

4. The vaccine according to any one of claims 1 to 3, wherein the fusion protein is a homodimer or heterodimer fusion protein, and optionally the fusion protein may further comprise one or more Th cell helper epitopes and / or connecting fragments in any one or both chains (i.e., the first polypeptide chain and / or the second polypeptide chain) of the homodimer or heterodimer. Optionally, the homodimeric fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are identical, for example, the first polypeptide chain and the second polypeptide chain sequentially comprise IFN, a target antigen, and an immunoglobulin Fc region (or Ab) from N-terminus to C-terminus, or a polypeptide of any combination order of the three structural units, and form a homodimer; preferably, the first polypeptide chain and the second polypeptide chain sequentially comprise IFN, a target antigen, and an immunoglobulin Fc region (or Ab) from N-terminus to C-terminus; it may also comprise a fusion protein of a Th cell helper epitope; Optionally, the heterodimeric fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are different, for example, the first polypeptide chain may include IFN and an immunoglobulin Fc region (or Ab) in sequence from the N-terminus to the C-terminus, or may include an immunoglobulin Fc region (or Ab) and IFN in sequence from the N-terminus to the C-terminus, and the second polypeptide chain may include a target antigen and an immunoglobulin Fc region (or Ab), wherein the target antigen may be located at the N-terminus and the immunoglobulin region (or Ab) may be located at the C-terminus, or the immunoglobulin region (or Ab) may be located at the N-terminus and the target antigen may be located at the C-terminus; or a polypeptide having any combination order of the three structural units to generate a heterodimer; preferably, the IFN and target antigenic sites are located at the N-termini of the two polypeptides, respectively, and the immunoglobulin Fc region (or Ab) is located at the C-termini of the two polypeptides; it may also include a fusion protein of a Th cell helper epitope.

5. The vaccine according to claim 4, wherein 1) The first polypeptide and the second polypeptide of the homologous dimer may comprise the amino acid sequences shown in SEQ ID NO. 11, 12, 13, 14, 29, 30, 31, 32, 38, 39, 40, 47, 48, 49, 50, 51, 56, 57, 59, 58, 65, 66, 67, and 68, 2) The first polypeptide of the heterodimer may comprise the nucleotide sequence of SEQ ID NOs. 15, 33, 42, 51, 60, and 69, and the second polypeptide may comprise the amino acid sequence of SEQ ID NOs. 16, 17, 18, 19, 34, 35, 36, 37, 43, 44, 45, 46, 52, 53, 54, 55, 61, 62, 63, 64, 70, 71, 72, and 73, 3) The antibodies may include DC-targeting antibodies, immune checkpoint blocking antibodies, immune-activating antibodies, etc., for example, vaccines containing the amino acid sequences of anti-PD-L1 antibodies (SEQ ID NO. 20), anti-DEC205 antibodies, anti-CD80 / 86 antibodies, etc.

6. A nucleic acid molecule encoding the fusion protein in the vaccine according to any one of claims 1 to 5, an expression vector comprising the nucleic acid molecule, or a host cell, such as a eukaryotic cell, comprising the nucleic acid molecule or expression vector.

7. Use of the fusion protein in the vaccine according to any one of claims 1 to 5 in the preparation of a composition or kit, such as a pharmaceutical or immunogenic composition or kit, a recombinant microorganism or a cell line.

8. The use according to claim 7, wherein the composition or kit is used for the prevention or treatment of tumors or pathogens, such as the prevention or treatment of viruses or bacteria, and the virus can be HBV, HPV, EBV, influenza virus, HIV, coronavirus, such as SARS-COV, SARS-COV-2, MERS-CoV, for example, the composition or kit is used as a hepatitis B preventive or therapeutic vaccine, a HBV preventive or therapeutic vaccine, an influenza preventive or therapeutic vaccine, a SARS-COV2 preventive or therapeutic vaccine, an HPV preventive or therapeutic vaccine, an HPV-related tumor preventive or therapeutic vaccine, an EBV preventive or therapeutic vaccine, an EBV-related tumor preventive or therapeutic vaccine, or an HIV preventive or therapeutic vaccine.

9. The vaccine according to any one of claims 1 to 5 or the use according to claim 7 or 8, wherein the vaccine, the composition or the kit can be administered via immunization routes such as intramuscular, intravenous, transdermal, subcutaneous or nasal, wherein the vaccine, the composition or the kit may further comprise an adjuvant, and the adjuvant may comprise 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 1-5 or the use according to claim 7 or 8, wherein the vaccine can be used in combination with another preventive or therapeutic therapy, for example, the vaccine can be an HBV therapeutic vaccine, the HBV therapeutic vaccine can be used in combination with another preventive or therapeutic HBV therapy, for example, the HBV 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 HBV therapeutic vaccine can be combined with nucleoside or nucleotide analogs, for example, for the treatment of chronic hepatitis B virus infection, for example, for the combined use of influenza, SARS-COV2, HPV, EBV, HIV preventive or therapeutic vaccines, etc. with antiviral drugs and other treatment methods; the combined use of HPV, EBV-related tumor preventive or therapeutic vaccines with antiviral and anti-tumor drugs and therapies, for example, any one of the vaccines according to any one of claims 1-5 as a component of a vaccine in a multivalent combination vaccine composed of other viruses or pathogens or tumor vaccines. For example, any multivalent vaccine of the SARS-COV-2 vaccine according to any one of claims 1-5 combined with an influenza vaccine or other vaccine, for example, any vaccine according to any one of claims 1-5 and an adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine for the same virus, pathogen, or tumor are immunized in a sequential or simultaneous immunization program, for example, the SARS-COV-2 fusion protein vaccine and the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine for SARS-COV-2 are immunized in a sequential or simultaneous immunization program, for example, the sequential immunization order can be: 1) first immunize with the SARS-COV-2 fusion protein vaccine of the present invention, and then immunize; 2) first immunize with the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine for SARS-COV-2, and then immunize with the ARS-COV-2 fusion protein vaccine; 3) immunize with the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine for SARS-COV-2 simultaneously.

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