Construction and application of fusion protein vaccine platform

Abstract

The present invention relates to the construction and application of a fusion protein vaccine platform. The present invention provides a vaccine comprising a fusion protein of an interferon-target antigen-immunoglobulin Fc region (or antibody) and a Th cell helper epitope. The present invention also relates to the use of a fusion protein containing an interferon-target antigen-immunoglobulin Fc (or antibody) region and a Th cell helper epitope for the preparation of a preventive or therapeutic composition. The vaccine of the present invention can be produced by a eukaryotic cell expression system to prepare vaccines of wild-type and various mutant antigens, and can be inoculated through immunization routes such as subcutaneous / muscular or nasal administration, which can induce a strong immune response in the body. The vaccine of the present invention can be used as a preventive or therapeutic vaccine.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and biomedical technology, and specifically relates to a vaccine, such as a fusion protein vaccine containing interferon-target antigen-immunoglobulin Fc region (antibody) as the main skeleton. The vaccine of the present invention can be used as a vaccine platform to prevent hepatitis B virus (HBV) infection, HPV, EBV, HIV, SARA-CoV-2, influenza virus infection and HPV, EBV related tumors, as well as to treat chronic hepatitis B (CHB) infection and HBV, HPV, EBV related tumors. Background Art

[0002] There are approximately 257 million people with chronic hepatitis B virus infection worldwide, and approximately 88,700 people die each year from end-stage liver disease caused by HBV, including liver failure, cirrhosis, and hepatocellular carcinoma. [1-3] About 30% of liver cirrhosis patients are caused by HBV, and about 40% of hepatocellular carcinoma (HCC) are caused by HBV. [4] . Hepatitis B virus infection remains a major global public health issue. However, there is still no effective strategy for the treatment of chronic hepatitis B. Existing HBV treatments mainly include antiviral drugs (nucleoside / nucleotide analogs) and interferon. Although they have certain therapeutic effects, they usually cannot induce effective immune responses and thus cannot completely eliminate HBV infection. Moreover, long-term medication causes significant side effects, and antiviral drugs can also develop drug resistance. Chronic HBV infection is one of the major diseases that threaten human health. It is urgent to explore effective immunotherapy strategies for chronic hepatitis B. The development of a therapeutic vaccine for chronic hepatitis B has very important social and economic significance.

[0003] Seasonal influenza causes severe illness in 1-4 million people and 200,000-500,000 deaths each year. [5] . 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 for influenza complications. Even though currently approved influenza vaccines can produce good protection in healthy young adults, there are still some problems that need to be solved. For example, some vaccines rely on chicken embryos for production, such as inactivated influenza vaccines and attenuated influenza vaccines. One disadvantage of these vaccines is that if the circulating strains are of avian origin, the epidemic will lead to an increase in demand for vaccines and chicken embryos, which will cause problems with the supply of chicken embryos. [6]Another disadvantage is that these vaccines take a long time to produce. Older people are more susceptible to severe syndromes from influenza viruses, and standard vaccines are generally less effective in older people, whose immune systems weaken with age. [7] 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.

[0004] When the antigen is linked to the Fc region of an immunoglobulin, the half-life of the antigen will be significantly prolonged, and the Fc region of the immunoglobulin can bind to the Fc receptor on the surface of antigen-presenting cells, thereby promoting the processing and presentation of the antigen by the antigen-presenting cells. [9-11] As an antiviral cytokine, type I interferon has many biological activities, one of which includes the stimulation of immune cells.

[12] IFNα can strongly induce the differentiation and activation of human DC cells

[13] After acting on immature DCs, type I interferons can promote the expression of MHC molecules and co-stimulatory molecules on the surface of DCs, such as MHC class I, CD80 and CD86, thereby enhancing the ability of DCs to activate T cells. [14-16] It has been reported that type I interferons can promote the antigen presentation ability of DCs after infection with vaccinia virus and lymphocytic chorioencephalitis virus (LCMV). [17-19] In addition, type I interferon can upregulate the expression of chemokine receptors after acting on DCs, promote the migration of DCs to lymph nodes, and then promote the activation of T cells. [20, 21] . 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

[22] The authors also found that the main cell group where type I interferon works is DC cells. At the same time, using antibodies to deliver the vaccine to DCs in a targeted manner, stimulating DC activation and cross-presentation function, will further enhance the activity and potency of the vaccine.

[0005] 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

[0006] Vaccines are an effective way to prevent and control major infectious diseases. There are various types of vaccines, one 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 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 proposed that the fusion of Th cell helper epitopes can further enhance the immune response effect of type I interferon-protein antigen-immunoglobulin Fc vaccine, which is an important component of the vaccine. The inventors proposed that antibodies such as anti-PD-L1 can be used to replace Fc, target the vaccine to DC, stimulate DC activation and cross-presentation function, and further enhance the activity and potency of the vaccine. As a new vaccine platform, the present invention can be used as a preventive and therapeutic vaccine for diseases such as viral infection, bacterial infection or tumor.

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

[0008] In some embodiments, the target antigen described herein is not particularly limited and can be any appropriate antigen. In some embodiments, the target antigen described herein can be, for example, a 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 in tumor cells, for example, human epidermal growth factor receptor 2 (HER2 / neu), epidermal growth factor (EGFR).

[0009] In some embodiments, the target antigen used in the vaccine provided by the present invention may be, for example, a mutated target antigen different from the wild type. In some embodiments, the target antigen described herein may be, for example, a mutant of a tumor antigen and / or a pathogen antigen (such as a viral or bacterial antigen). In some embodiments, the target antigen may be, for example, the full length or S1 region of the SARS-COV-2 virus S protein, for example, the target antigen may be an antigen shown 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 encoded by a wild-type gene (a wild-type gene refers to the majority allele in nature, often used as a standard control gene in biological experiments), 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 mutation. For example, the point mutations of different mutant coronavirus S proteins 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 mutant coronaviruses derived from the British B.1.1.7 (Alpha) mutant, the South African B.1.351 (Beta) mutant, the Brazilian P1 (Gamma) mutant, the Indian B.1.617, B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.617.3 mutant, the California B.1.429 mutant, etc. In some embodiments, the mutated target antigen may include, for example, natural point mutations / deletion mutations / increase mutations / truncations, artificial point mutations / deletion mutations / increase mutations / truncations, any combination of natural or artificial mutations, and subtypes produced after mutation, 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 / increase mutations / truncations of SARS-COV-2 proteins (for example, one or more of the S protein, N protein, M protein, and E protein), artificial point mutations / deletion mutations / increase 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, the S1 region, or the RBD region, 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 mutations, L452R mutations, T478K mutations, D614G, H655Y mutations, for example, the mutated viral antigen may include the B.1.1.7 (Alpha) mutant strain from the UK, the B.1.351 (Beta) mutant strain from South Africa, 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 include 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. In this article, unless otherwise explicitly stated or clearly limited by the context, the target antigen mentioned herein generally includes wild-type target antigens and mutant target antigens.

[0010] The present invention aims to provide a vaccine platform, which is composed of 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 fragment, herpes zoster virus (VZV) gE antigen, Epstein-Barr virus (EBV) EBNA1 / LMP2 / gp350, herpes simplex virus 2 (HSV-2) gD Antigen, HIV gp120 antigen-immunoglobulin Fc region (or antibody) composition (additional Th epitope). The fusion protein can be a homologous or heterologous dimeric protein. When the fusion protein is a dimer, the interferon, target antigen, immunoglobulin Fc region (or antibody Ab) as structural units can exist in the first polypeptide chain and / or the second polypeptide chain, and the existence of each structural unit is not particularly limited, for example, it can exist in one chain at the same time, or any one or more structural units can exist in one chain, and another one or more structural units can exist in another chain.

[0011] The interferon of the present invention can be selected from type I interferon, type II interferon and type III interferon, such as IFN-α, IFN-β, IFN-γ, IFN-λ1 (IL-29), IFN-λ2 (IL-28a), IFN-λ (IL-28b) and IFN-ω; the IFN can be from human or mouse sources; preferably type I interferon IFN-α (SEQ ID NO.1, SEQ ID NO.21, SEQ ID NO.22).

[0012] The immunoglobulin Fc region of the present invention can be selected from the constant region amino acid sequence of IgG1, IgG2, IgG3 and IgG4 / or IgM, preferably IgG1 (SEQ ID NO.2, SEQ ID NO.23, SEQ ID NO.24).

[0013] The fusion polypeptide of the present invention may also optionally contain one or more Th cell helper epitopes and / or connecting fragments (linkers). For example, when the fusion protein is a dimer, the fusion protein may optionally contain one or more Th cell helper epitopes and / or connecting fragments in any one or two chains (i.e., the first polypeptide chain and / or the second polypeptide chain) of the homodimer or heterodimer. As known to those skilled in the art, the various structural units of the fusion protein may be connected by appropriate connecting fragments (linkers). The connecting fragments that can be used in the vaccine of the present invention are not particularly limited and may be any appropriate peptide fragment known in the art. The connecting fragments of the various structural units of the present invention may be flexible polypeptide sequences, and may be connecting fragments 1 and 2, such as shown in the amino acid sequences of SEQ ID NO.4 and SEQ ID NO.25.

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

[0015] The preferred antigens described in the present invention include hepatitis B Pres1 antigen, including ad subtype (SEQ ID NO.6) and ay subtype (SEQ ID NO.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), and HPV E7 antigen (SEQ ID NO.10). Herpes virus VZV-gE antigen (SEQ ID NO.91), EBV-gp350 antigen (SEQ ID NO.92), and HSV-2-gD antigen (SEQ ID NO.93).

[0016] The homodimeric protein described in the present invention comprises a first polypeptide and a second polypeptide, and the first polypeptide is identical to the second polypeptide. The order of the first polypeptide and the second polypeptide from segment N to segment C is IFN-tumor or viral antigen (hepatitis B Pres1 antigen, SARS-COV2 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. It comprises the amino acid sequence 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, 68.

[0017] The heterodimer of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same polypeptide, the first polypeptide is respectively an IFN-immunoglobulin Fc region from the C segment to the N-terminus, comprising the amino acid sequences shown in SEQ ID NO.15, 33, 42, 51, 60, and 69; the second polypeptide is respectively a tumor or virus 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 segment; 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.

[0018] 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.

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

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

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

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

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

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

[0025] The present invention further includes the application of the vaccine platform, which can be used as a hepatitis B preventive vaccine, the vaccine platform as a hepatitis B therapeutic vaccine, the vaccine platform as an influenza preventive vaccine, the vaccine platform as a SARA-COV2, influenza, HPV, VZV, EBV, HSV-2, HIV preventive vaccine, and the vaccine platform as a HPV, EBV related tumor preventive vaccine.

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

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

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

[0029] The present invention includes the combined use of the vaccine platform as a preventive or therapeutic vaccine for HBV, influenza, SARA-COV2, HPV, VZV, EBV, HSV-2, HIV, etc. with antiviral drugs and other treatment methods; and the combined use of HBV, HPV, EBV-related tumor preventive or therapeutic vaccines with antiviral and anti-tumor drugs and therapies.

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

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

[0032] The present invention comprises 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.

[0033] The present invention comprises any possible mutants of the fusion protein vaccine antigen, including natural point mutations / deletion mutations / truncations, any combination of natural point mutations, subtypes produced after mutation, and artificial point mutations / deletion mutations / truncations and other mutant sequences 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.

[0034] 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 of the SARS-COV-2 fusion protein vaccine of the present invention combined with 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, 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 Or DNA vaccine is immunized in a sequential or simultaneous immunization program, for example, the sequential immunization order can be: 1) first immunize the SARS-COV-2 fusion protein vaccine of the present invention, then immunize the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of SARS-COV-2; 2) first immunize the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of SARS-COV-2, then immunize the ARS-COV-2 fusion protein vaccine; 3) SARS-COV-2 fusion protein vaccine and adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of SARS-COV-2 are immunized simultaneously. As known in the art, when used in combination, the vaccines used in combination can be prepared as a convenient kit.

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

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

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

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

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

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

[0041] 6. The IFN-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.

[0042] 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.

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

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

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

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

[0047] Sequence information involved in this article:

[0048] 1. Unit component sequence:

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

[0050] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKE

[0051] SEQ ID NO.21: Human IFNα2 amino acid sequence (hIFNα)

[0052] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKE

[0053] SEQ ID.NO.22: Human mutant IFNα2 (Q124R) amino acid sequence (hmIFNα)

[0054] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE

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

[0056] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV

[0057] SEQ ID No.23: Heterodimeric Fc-hole

[0058] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0059] SEQ ID No.24: Heterodimeric Fc-knob

[0060] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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

[0062] AKFVAAWTLKAAA

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

[0064] GGGGSGGGGSGGGGS

[0065] SEQ ID NO.25: Linker 2 amino acid sequence:

[0066] GSGSGS

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

[0068] MARLCAFLMILVMMSYYWSACSLG

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

[0070] MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVSTIPPPASTNRQSGRQPTPISPPLRDSHPQA

[0071] SEQ ID NO.26: Amino acid sequence of HBV Pres1 (ay subtype)

[0072] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQA

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

[0074] MENTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYNILSPFLPLLPIFFCLWVYI

[0075] SEQ ID NO.27: Amino acid sequence of HBV HBsAg (adw subtype)

[0076] MENITSGLLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLSFLGEAPVCLGQNSQSPTRNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMIWYWGPSLYSIVCPFTPLLQIFCCLWVFI

[0077] SEQ ID NO.28: Amino acid sequence of HBV HBsAg (ayw subtype)

[0078] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQSSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI

[0079] SEQ ID NO.8: Amino acid sequence of SARS-CoV-2 RBD

[0080] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0081] SEQ ID NO.9: Amino acid sequence of HA of influenza virus

[0082] DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSR

[0083] SEQ ID NO.10: Amino acid sequence of HPV-E7 antigen

[0084] MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP

[0085] 2. Mouse IFN vaccine mIFNα - antigen - Fc sequence:

[0086] SEQ ID NO.11: Amino acid sequence of mIFNα - Pres1 - Fc in a homodimer

[0087] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV

[0088] SEQ ID NO.12: Amino acid sequence of mIFNα - RBD (SARS-CoV-2) - Fc in a homodimer

[0089] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV

[0090] SEQ ID NO.13: Amino acid sequence of mIFNα-HA-Fc in the homodimer

[0091] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV

[0092] SEQ ID NO.14: Amino acid sequence of mIFNα - E7 (HPV)-Fc in a homodimer

[0093] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKHV

[0094] SEQ ID NO.15: Amino acid sequence of the first chain mIFNα-Fc-hole in the heterodimer

[0095] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0096] SEQ ID NO.16: Amino acid sequence of the second chain Pres1-Fc-knob in the heterodimeric mIFNα-Pres1-Fc

[0097] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0098] 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

[0099] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0100] SEQ ID NO.18: Amino acid sequence of the second chain HA-Fc-knob in the heterodimeric mIFNα-HA-Fc

[0101] DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0102] SEQ ID NO.19: Amino acid sequence of the second chain E7-Fc-knob in the heterodimeric mIFNα-E7 (HPV)-Fc

[0103] MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0104] 3. Mouse IFN vaccine containing Pan epitope IFNα-Pan-antigen-Fc sequence:

[0105] SEQ ID NO.29: Amino acid sequence of mIFNα-Pan-Pres1-Fc in homodimer

[0106] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSL CAWEVIRAEVWRALSSSTNLLARLSEEKEGGGGSGGGGSGGGGSRTAKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQ AQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0107] SEQ ID NO.30: Amino acid sequence of mIFNα-Pan-RBD (SARS-CoV-2)-Fc in homodimer

[0108] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKEGGGGSGGGGSGGGGSRTAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0109] SEQ ID NO.31: Amino acid sequence of mIFNα-Pan-HA-Fc in the homodimer

[0110] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKEGGGGSGGGGSGGGGSRTAKFVAAWTLKAAAGSGSGSDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0111] SEQ ID NO.32: Amino acid sequence of mIFNα-Pan-E7 (HPV)-Fc in a homodimer

[0112] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKEGGGGSGGGGSGGGGSRTAKFVAAWTLKAAAGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0113] SEQ ID NO.33: Amino acid sequence of the first chain mIFNα-Fc-hole in the heterodimer

[0114] CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0115] SEQ ID NO.34: Amino acid sequence of the second chain Pan-Pres1-Fc-knob in the heterodimeric mIFN-Pan-Pres1-Fc

[0116] AKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0117] 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

[0118] AKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0119] SEQ ID NO.36: Amino acid sequence of the second chain Pan-HA-Fc-knob in the heterodimeric mIFNα-Pan-HA-Fc

[0120] AKFVAAWTLKAAAGSGSGSDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0121] SEQ ID NO.37: Amino acid sequence of the second chain Pan-E7-Fc-knob in the heterodimeric mIFNα-Pan-E7 (HPV)-Fc

[0122] AKFVAAWTLKAAAGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0123] 4. Human IFN vaccine hIFNα-antigen-Fc sequence:

[0124] SEQ ID NO.38: Amino acid sequence of hIFNα-Pres1-Fc in homodimer

[0125] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0126] SEQ ID NO.39: Amino acid sequence of hIFNα-RBD (SARS-CoV-2)-Fc in a homodimer

[0127] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0128] SEQ ID NO.40: Amino acid sequence of hIFNα-HA-Fc in the homodimer

[0129] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0130] SEQ ID NO.41: Amino acid sequence of hIFNα-E7 (HPV)-Fc in a homodimer,

[0131] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0132] SEQ ID NO.42: Amino acid sequence of the first chain hIFN-Fc-hole in the heterodimer

[0133] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFKLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0134] SEQ ID NO.43: Amino acid sequence of the second chain Pres1-Fc-knob in the heterodimer hIFNα-Pres1-Fc

[0135] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0136] 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

[0137] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0138] SEQ ID NO.45: Amino acid sequence of the second chain HA-Fc-knob in the heterodimer hIFNα-HA-Fc

[0139] DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0140] SEQ ID NO.46: Amino acid sequence of the second chain E7(HPV)-Fc-knob in the heterodimeric hIFNα-E7 (HPV)-Fc

[0141] MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0142] 5. Human IFN vaccine containing Pan epitope IFNα-Pan-antigen-Fc sequence:

[0143] SEQ ID NO.47: Amino acid sequence of hIFNα-Pan-Pres1-Fc in homodimer

[0144] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYL KEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQT LPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0145] SEQ ID NO.48: hIFNα-Pan-RBD (SARS-CoV-2)-Fc amino acid sequence in homodimer

[0146] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0147] SEQ ID NO.49: Amino acid sequence of hIFNα - Pan - HA - Fc in the homodimer

[0148] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0149] SEQ ID NO.50: Amino acids of hIFNα-Pan-E7 (HPV)-Fc in the homodimer

[0150] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0151] SEQ ID NO.51: Amino acid sequence of the first chain hIFNα-Fc-hole in the heterodimer

[0152] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0153] SEQ ID NO.52: Amino acid sequence of the second chain Pan-Pres1-Fc-knob in the heterodimer hIFNα-Pan-Pres1-Fc

[0154] AKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0155] 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

[0156] AKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0157] SEQ ID NO.54: Amino acid sequence of the second chain Pan-HA-Fc-knob in the heterodimer hIFNα-Pan-HA-Fc

[0158] AKFVAAWTLKAAAGSGSGSDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0159] SEQ ID NO.55: Amino acid sequence of the second chain Pan-HA-Fc-knob in the heterodimer hIFNα-Pan-E7(HPV)-Fc

[0160] AKFVAAWTLKAAAGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0161] 6. Human mutant IFN vaccine hmIFNα-Pan-antigen-Fc sequence:

[0162] SEQ ID NO.56: Amino acid sequence of hmIFNα-Pres1-Fc in homodimer

[0163] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0164] SEQ ID NO.57: Amino acid sequence of hmIFNα-RBD (SARS-CoV-2)-Fc in a homodimer

[0165] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0166] SEQ ID NO.58: Amino acid sequence of hmIFNα-HA-Fc in a homodimer

[0167] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0168] SEQ ID NO.59: Amino acid sequence of hmIFNα - E7 (HPV)-Fc in a homodimer

[0169] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSRTMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0170] SEQ ID NO.60: Amino acid sequence of the first chain hmIFN-Fc-hole in the heterodimer

[0171] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0172] SEQ ID NO.61: Amino acid sequence of the second chain Pres1-Fc-knob in the heterodimer hmIFNα-Pres1-Fc

[0173] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0174] 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

[0175] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0176] SEQ ID NO.63: Amino acid sequence of the second chain HA-Fc-knob in the heterodimer hmIFNα-HA-Fc

[0177] DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0178] SEQ ID NO.64: Amino acid sequence of the second chain HA-Fc-knob in the heterodimeric hmIFNα-E7 (HPV)-Fc

[0179] MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0180] 7. Human mutant IFN containing Pan epitope vaccine hmIFNα-Pan epitope-antigen-Fc sequence

[0181] SEQ ID NO.65: Amino acid sequence of hmIFNα-Pan-Pres1-Fc in homodimer

[0182] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYL KEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQT LPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0183] SEQ ID NO.66: Amino acid sequence of hmIFNα-Pan-RBD (SARS-CoV-2)-Fc in homodimer

[0184] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0185] SEQ ID NO.67: Amino acid sequence of hmIFNα-Pan-HA-Fc in a homodimer

[0186] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0187] SEQ ID NO.68: Amino acid sequence of hmIFNα-Pan-E7 (HPV)-Fc in a homodimer

[0188] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAKFVAAWTLKAAAGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0189] SEQ ID NO.69: Amino acid sequence of the first chain hmIFNα4-Fc-hole in the heterodimer

[0190] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESGGGGSGGGGSGGGGSGGGGRTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0191] SEQ ID NO.70: Amino acid sequence of the second chain Pan-Pres1-Fc-knob in the heterodimer hmIFNα-Pan-Pres1-Fc

[0192] AKFVAAWTLKAAAGSGSGSMGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQTGRQPTPLSPPLRNTHPQAFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0193] 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

[0194] AKFVAAWTLKAAAGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0195] SEQ ID NO.72: Amino acid sequence of the second chain Pan-HA-Fc-knob in the heterodimer hmIFNα-Pan-HA-Fc

[0196] AKFVAAWTLKAAAGSGSGSDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0197] SEQ ID NO.73: Amino acid sequence of the second chain Pan-HA-Fc-knob in the heterodimer hmIFNα-Pan-E7 (HPV)-Fc

[0198] AKFVAAWTLKAAAGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPFEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0199] 8. Antibody sequence substituting for Fc

[0200] SEQ ID NO.20: Amino acid sequence of ScFv(PD-L1)

[0201] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA

[0202] SEQ ID NO.74: Amino acid sequence of Anti-PD-L1 VH

[0203] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA

[0204] SEQ ID NO.75: Amino acid sequence of Anti-PD-L1 VL

[0205] DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR

[0206] 9. Other viral antigen sequences

[0207] SEQ ID NO. 76 Amino acid sequence of SARS-CoV-2 Spike protein

[0208]

[0209] Amino acid sequence of SEQ ID NO. 77 SARS-CoV-2 S1 protein

[0210] VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR

[0211] Amino acid sequence of SEQ ID NO. 78 SARS-CoV-2 wild-type strain RBD protein

[0212] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0213] 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)

[0214] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0215] 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)

[0216] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0217] SEQ ID NO. 81 Amino acid sequence of the RBD protein of the Brazilian mutant strain (P.1) of SARS-CoV-2

[0218] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGTIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0219] SEQ ID NO. 82 Amino acid sequence of the RBD protein of the California mutant strain of SRAS-CoV-2 (B.1.429)

[0220] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0221] 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

[0222] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVQGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0223] SEQ ID NO. 84 Amino acid sequence of the RBD protein of the second generation B.1.617.2 (Delta) mutant strain of SRAS-CoV-2 from India

[0224] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0225] 10. Other tumor antigen sequences:

[0226] The amino acid sequences of the mouse Her2 extracellular domains II, III and IV involved in the examples are:

[0227] SEQ ID NO. 85 Mouse Her2-extracellular domain 2:

[0228] SRACPPCAPACKDNHCWGESPEDCQILTGTICTSGCARCKGRLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMHNPEGRYTFGASCVTTCPYNYLSTEVGSCTLVCPPNNQEVTAEDGTQRCEKCSKPC

[0229] SEQ ID NO. 86 Mouse Her2-extracellular domain 3:

[0230] GCKKIFGSLAFLPESFDGDPSSGIAPLRPEQLQVFETLEEITGYLYISAWPDSLRRDLSVFQNLRIIRGRILHDGAYSLTLQGLGIHSLGLRSLRELGSGLALIHRNAHLCFVHTVPWDQLF

[0231] SEQ ID NO. 87 Mouse Her2-extracellular domain 4:

[0232] VCNSLCAHGHCWGPGPTQCVNCSHFLRGQECVEECRVWKGLPREYVSDKRCLPCHPECQPQNSSETCFGSEADQCAACAHYKDSSSCVARCPSGVKPDLSYMPIWKYPDEEGICQPCPINCTHSCVDLDERGCP

[0233] The amino acid sequences of the human Her2 extracellular domains II, III and IV involved in the examples are:

[0234] SEQ ID NO. 88 Human Her2-extracellular domain 2:

[0235] SRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPC

[0236] SEQ ID NO. 89 Human Her2-extracellular domain 3:

[0237] GCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPW

[0238] SEQ ID NO. 90 Human Her2-extracellular domain 4:

[0239] CHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCP

[0240] 11. Herpes virus antigen sequences involved in the examples:

[0241] SEQ ID NO. 91 VZV Envelope glycoprotein E (aa 31-538)

[0242] SVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRY

[0243] SEQ ID NO. 92 EBV Envelope glycoprotein GP350 (aa 1-425)

[0244] MEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCTADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALTM RSKKLPINVTTGEEQQVSLESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNCNSTNITAVVRAQGLDVTLPLSLPTSAQDSNFSVKTQMLGNEIDIECI MEDGEISQVLPGDNKFNITCSGYESHVPSGGILTSTSPVVTPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKASGGDYCIQSNIVFSDEIPASQDMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWAWPNNTETDFKCKWTLTSGTPSGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTTHKVIFSKAP

[0245] SEQ ID NO. 93 HSV-2 Envelope glycoprotein gD (aa 26-339)

[0246] KYALADPSLKMADPNRFRGKNLPVLDRLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPA FETAGTYLRLVKINDWTEITQFILEHRRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNP BRIEF DESCRIPTION OF THE DRAWINGS

[0247] 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);

[0248] Figure 2. Schematic diagram of the vaccine platform in the form of a heterodimer, with the combination of interferon-linker fragment 1-IgG1-hole and target antigen-IgG1-knob (or antibody);

[0249] Figure 3 . The vaccine platform is in the form of a heterodimer, according to the schematic diagram of the interferon-linker fragment 1-IgG1-knob combination and the target protein-IgG1-hole (or antibody);

[0250] 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);

[0251] Figure 5 . Schematic diagram of the vaccine platform in the form of heterodimers, respectively according to the combination of interferon-linking fragment 1-IgG1-hole and Th cell helper epitope-linking fragment 2-target antigen-IgG1-knob (or antibody);

[0252] Figure 6 . Schematic diagram of the vaccine platform in the form of heterodimers, respectively according to the combination of interferon-linking fragment 1-IgG1-knob and Th cell helper epitope-linking fragment 2-target antigen-IgG1-hole (or antibody).

[0253] Figure 7 . SDS-PAGE electrophoresis identification of Pres1-Fc and IFN-Pres1-Fc non-denatured proteins

[0254] Figure 8 Compared with free preS1, fusion proteins preS1-Fc and IFN-preS1-Fc can significantly enhance the immunogenicity of antigen molecules and induce the production of broad-spectrum neutralizing antibodies. (a) C57 / BL6 mice (n=8 / group) were subcutaneously immunized with free HBV Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins, and the levels of Pres1-specific antibodies in serum were detected by Elisa at the specified time. (b) Mice (n=4) stably carrying the three HBV genotypes were injected intravenously with serum from mice immunized with IFNα-Pres1-Fc protein, and the changes in Pres1 antigen in serum were detected 12 hours later.

[0255] Fig. 9IFNα-Pres1-Fc can be used as a preventive vaccine for hepatitis B. C57 / BL6 mice were subcutaneously immunized with free hepatitis B Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins. On day 28 of vaccination, 1x10 11 vg of AAV-HBV1.3 virus. (a) Serum Anti-Pres1 levels before virus inoculation and at 1, 2, 3, and 4 weeks after virus inoculation. (b) Serum Pres1 levels were detected at designated time points. (c) Serum HBsAg levels were detected at 1, 2, 3, and 4 weeks by Elisa. (d) The proportion of HBsAg-positive mice after AAV-HBV1.3 virus inoculation.

[0256] Fig.10 IFNα-Pres1-Fc as a therapeutic vaccine for chronic hepatitis B infection. C57 / BL6 mice were infected with 1x10 11 vg of AAV-HBV1.3 virus, and stably infected mice were selected after 6 weeks of infection (n=8 / group), and recombinant Pres1 and IFNα-Pres1-Fc proteins were inoculated subcutaneously, once every 2 weeks for a total of three immunizations. (a) Detection of Anti-Pres1 antigen in serum; (b) Detection of Pres1 antigen in serum; (c) Detection of HBV-related antigen HBsAg level in mouse serum

[0257] Fig.11 Th cell helper epitopes enhance antibody responses to IFNα-Pres1-Fc vaccine

[0258] Compared with IFN-preS1-Fc, IFN-Pan-preS1-Fc can significantly enhance the immunogenicity of the antigen molecule. C57 / BL6 (n=8 / group) mice were subcutaneously immunized with hepatitis B Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins without aluminum adjuvant, and the level of Pres1-specific antibodies in serum was detected by Elisa at the specified time.

[0259] Fig.12 IFNα-Pan-Pres1-Fc as a therapeutic vaccine for chronic hepatitis B infection. C57 / BL6 mice were infected with 1x10 11vg of AAV-HBV1.3 virus, and stably infected mice (n=8 / group) were selected after 6 weeks of infection. Recombinant Pres1 and IFNα-Pres1-Fc proteins were inoculated subcutaneously, and immunized once every 2 weeks for a total of three times. (a) Detection of Anti-Pres1 antigen in serum; (b) Detection of Pres1 antigen in serum; (c) Detection of HBV-related antigen HBsAg level in mouse serum; (d) Detection of HBV-DNA level in mouse serum by QPCR.

[0260] Fig.13 IFNα-Pres1-Fc combined with HBsAg commercial vaccine breaks the immune tolerance caused by HBsAg and induces HBsAg-HBsAb serological conversion. HBV Carrier mice were subcutaneously immunized with IFNα-Pres1-Fc and HBsAg commercial vaccine, once every two weeks for a total of three immunizations. (a) The level of Pres1 in the serum of HBV Carrier mice, (b) the level of HBsAg, (c) the level of Anti-Pres1 in the serum, (d) the level of Anti-HBsAg in the serum, and (e) the level of HBV-DNA in the serum. ***, p<0.001

[0261] Fig.14 . IFNα-RBD (SARS-CoV2)-Fc can induce stronger antibody response than free SARS-Cov2 RBD protein. Balb / c mice (n=8 / group) were subcutaneously immunized with free SARS-Cov-2 RBD, RBD-Fc, and IFNα-RBD-Fc proteins, and the levels of SARS-Cov2 S protein-specific antibodies in serum were detected by Elisa at the specified time. ****, p<0.0001.

[0262] Fig.15 . IFNα-RBD (SARS-CoV2)-Fc produced high-titer antiviral serum after immunization of mice, which could completely block SARS-CoV2 pseudovirus infection in in vitro cell experiments.

[0263] Fig.16. Detection of anti-serum RBD-specific antibodies produced by IFNα-Pan-RBD (original strain)-Fc and IFNα-Pan-RBD (SARS-CoV-2 South Africa mutant strain)-Fc immunization. (a) IFNα-Pan-RBD (SARS-CoV-2 original strain)-Fc SDS-PAGE electrophoresis identification diagram. (b) IFNα-Pan-RBD (SARS-CoV-2 South Africa mutant strain)-Fc SDS-PAGE electrophoresis identification diagram. (c) Binding of RBD-specific antibodies to the original strain RBD in serum 14 days after IFNα-Pan-RBD (original strain)-Fc and IFNα-Pan-RBD (SARS-CoV-2 South Africa mutant strain)-Fc immunization. (d) Binding of RBD-specific antibodies to the South African mutant RBD in the serum 14 days after immunization with IFNα-Pan-RBD (original strain)-Fc and IFNα-Pan-RBD (SARS-CoV-2 South African mutant)-Fc.

[0264] Fig.17 (a) SDS-PAGE electrophoresis identification of Mouse IFNα-RBD-Fc and Mouse IFNα-Pan-RBD-Fc proteins. (b) SDS-PAGE electrophoresis identification of Human IFNα-RBD-Fc and Human IFNα-Pan-RBD-Fc proteins.

[0265] Fig.18 . Pan (Pan DR-binding epitope) CD4 T cell helper epitope can further enhance the immunogenicity of IFNα-RBD-Fc. Mice were inoculated with 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 protein by intramuscular immunization, and a booster immunization was performed 14 days after inoculation. Mouse sera were collected on days 7, 14, and 28 after immunization, and the levels of RBD-specific antibodies in mouse sera were detected by ELISA. *, p<0.05; ****, p<0.0001.

[0266] Fig.19. Aluminum adjuvant can enhance the specific humoral immune response induced by Human IFNα-RBD-Fc and Human IFNα-Pan-RBD-Fc proteins. C57BL / 6 mice were vaccinated with 10 μg Human IFNα-RBD-Fc or Human IFNα-Pan-RBD-Fc protein on days 0 and 14 with or without aluminum adjuvant (AL+). The mouse sera were collected on days 7, 14, and 28 after vaccination, and the SARS-CoV-2 RBD-specific antibody levels in the mouse sera were detected by ELISA. *, p<0.05; ****, p<0.0001.

[0267] Fig. 20 . Intranasal immunization with IFN-Pan-RBD-Fc can induce the production of high titer RBD-specific IgG and IgA neutralizing antibodies. 6-8 week old C57BL / 6 mice were divided into 5 groups, 10 mice in each group, and immunized with 10 μg of IFNα-pan-RBD-Fc or the same molar number of RBD, RBD-Fc, and IFNα-RBD-Fc proteins by intranasal immunization, with a nasal drop volume of 10uL / mouse. Mice were immunized twice on days 0 and 14. Mouse sera were collected on days 7, 14, 21, 28, 35, and 42 after immunization, and the levels of coronavirus RBD-specific antibodies IgG (a) and IgA (b) in the sera of each group were detected by ELISA; sera on day 42 were taken for in vitro neutralization experiments of SARS-CoV-2 pseudoviruses (c). Statistical method: one-way ANOVA, *p<0.05 means significant difference, **p<0.01, ***p<0.001, ****p<0.0001.

[0268] Fig.21 . Intranasal immunization with the unadjuvanted coronavirus vaccine IFN-Pan-RBD-Fc induced high titer RBD-specific IgG and IgA neutralizing antibodies in the nasopharynx and lung tissues. 6-8 week old C57BL / 6 mice were killed 28 days after immunization, and the nasal mucosa of the mice was obtained and broken with a tissue homogenizer. The homogenized liquid was centrifuged at 13000rpm for 10 minutes, and the supernatant was taken as the nasal mucosal supernatant (NMDS). The ELISA method was used to detect the levels of coronavirus RBD-specific antibodies IgG (a) and IgA (b) in the nasal mucosal supernatant (NMDS) of each group; the serum was taken 28 days later for in vitro neutralization experiment of SARS-CoV-2 pseudovirus (c). Statistical method: one-way ANOVA, *p<0.05 is significantly different, **p<0.01, ***p<0.001, ****p<0.0001.

[0269] Fig. 22 . Intranasal immunization with the unadjuvanted coronavirus 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. Fig.19 ) were killed. For the mouse lungs, use a 1ml syringe to draw about 0.8ml of HBSS+100uMEDTA, inject it into the endotracheal tube, gently blow and aspirate three times, then aspirate the liquid and collect it in a centrifuge tube. Repeat this step three times, and finally about 2ml of lung lavage fluid can be obtained. The mouse lung lavage fluid was centrifuged at 500g for 5 minutes, and the supernatant was the mouse lung lavage fluid (BALF). The ELISA method was used to detect the content of coronavirus RBD-specific antibodies IgG (a) and IgA (b) in the lung lavage fluid (BALF) of each group of mice. Statistical method: one-way ANOVA, *p<0.05 is significantly different, **p<0.01, ***p<0.001, ****p<0.0001.

[0270] Fig.23 . Expression and purification of Her2 vaccine proteins. The relevant proteins were expressed and purified in 293F cells, and the size and purity of the proteins were detected by SDS-PAGE and Coomassie Brilliant 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).

[0271] Fig.24 . Analyze the antitumor activity of Her2 vaccine IFNα-3-Fc and IFNα-pan-3-Fc. Construct TUBO breast cancer model mice, and treat the tumor size of 50-80mm3. Inject the relevant fusion protein into the tumor once a week for a total of 3 times. The dosage of IFNα-3-Fc is 10ug / / dose / mouse, and other fusion proteins are administered in equimolar amounts. CpG is used as an adjuvant. Measure the tumor size and draw a tumor growth curve.

[0272] Fig.25 . Analysis of the function of IFNα and Pan in improving the immunogenicity of Her2 antigen. BALB / C mice aged 6-8 weeks (n=5) were subcutaneously immunized with mouse Her2 fusion protein vaccines 4-Fc, IFNα-4-Fc and IFNα-pan-4-Fc without adding adjuvant. The immunization dose was IFNα-4-Fc 10ug / dose / / mouse, and other fusion proteins were inoculated in equal moles. Venous blood was collected 14 days and 21 days after immunization, and the antibody level of Her2-specific IgG in serum was detected by ELISA.

[0273] Fig.26 . SDS-PAGE electrophoresis identification pattern of IFN-HA1-Fc fusion protein.

[0274] Fig. 27 .Mice were inoculated with 10ug IFN-HA1-Fc or the same molar amount of HA1 protein by intramuscular immunization, and a booster immunization was performed 14 days after the initial vaccination. Mouse serum was collected on the 28th day after immunization, and the level of HA1-specific antibodies in the mouse serum was detected by ELISA. Mice were infected with 1000 PFU of A / PR8 influenza virus by nasal infection at 42 days after immunization. From the third day after virus infection, the mice were observed and their weight changes were recorded. (a) Mouse serum was collected 28 days after the initial immunization, and the level of HA1-specific antibodies in the mouse serum was detected by ELISA. (b) Weight changes of mice after virus infection.

[0275] 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.

[0276] Fig.29 . IFNα-Pan-EBV gp350-Fc can induce a stronger humoral immune response than free EBV gp350. C57BL / 6 (n=5 / group) mice were immunized with 10 μg IFNα-Pan-EBV gp350-Fc or the same molar amount of EBV gp350 protein by intramuscular inoculation, and boosted with the same dose 14 days after the primary immunization. The mouse sera were collected 14 and 28 days after the primary immunization, and the EBV gp350-specific antibody level in the mouse sera was detected by ELISA.

[0277] Fig.30 . IFNa-Pan-HSV-2 gD-Fc can induce a stronger humoral immune response than free HSV-2 gD protein. C57BL / 6 (n=5) mice were inoculated with 10 μg IFNa-Pan-HSV-2 gD-Fc or the same molar amount of HSV-2 gD protein by intramuscular immunization on days 0 and 14, respectively. The mouse sera were collected on days 14 and 28 after the initial inoculation, and the levels of HSV-2 gD-specific antibodies in the sera were detected by ELISA.

[0278] Fig.31. 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.

[0279] C57BL / 6 mice (n=6 / group) were immunized intramuscularly with 10 μg IFNα-Pan-VZV gE-Fc or the same molar amount of VZV E protein, and boosted with the same dose 14 days after the primary immunization. Mouse sera were collected 14 and 28 days after the primary immunization, and the EBV gp350-specific IgG level in the mouse sera was detected by ELISA (a). VZV gG-specific IgG1 (b) and IgG2c (c) antibody subtypes were detected in the mouse sera 28 days after the primary immunization. DETAILED DESCRIPTION

[0280] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be described in detail with reference to the embodiments and drawings below. The embodiments introduced are only examples of the present invention, but are not intended to limit the scope of the present invention. The examples are only a part of the present invention, but not all embodiments. The scope of the present invention is limited by the specific requirements of the attached claims.

[0281] Example 1. Design of vaccine platform

[0282] The vaccine platform of interferon-target antigen-immunoglobulin Fc (or antibody) structural unit is composed of three structural units. The first structural unit is the interferon part, the second structural unit is the immunoglobulin Fc region (or antibody), and the third unit is the target antigen. In actual construction, the three structural units can be arranged and combined in any form and the target antigen can be connected to the Th cell helper epitope through the connecting sequence 2. Its representative form is as follows:

[0283] 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.

[0284] 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.

[0285] 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.

[0286] 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:

[0287] 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.

[0288] 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.

[0289] 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.

[0290] Example 2. Construction, purification and production of vaccine platform

[0291] We describe the expression and production of this vaccine platform using the hepatitis B virus Pres1 and the coronavirus SARS-CoV-2 RBD protein homodimer as examples.

[0292] 1. Vector construction, host cell transfection and induced expression

[0293] 1.1. Using PEE12.4 as a vector, the vaccine structural unit was constructed into the vector by molecular cloning to obtain a plasmid that can express the fusion protein. The fusion protein was then transiently transfected into 293F cells, the culture supernatant was collected, and the target protein was finally purified by Protein A affinity chromatography.

[0294] Vector construction (taking HBV preS1 antigen as an example)

[0295] (1) PEE12.4-HindIII-Signal Peptide 1-Interferon-BsiwI-Pres1-BstbI-hIgG1-EcoRI

[0296] (2) PEE12.4-HindIII-Signal Peptide 1-Interferon-BsiwI-RBD (SARS-CoV-2)-BstbI-hIgG1-EcoRI

[0297] (3) PEE12.4-HindIII-Signal Peptide 1-Interferon-Bsiwi-PADER-Pres1-hIgG1-EcoRI

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

[0299] The connecting sequence between each fusion protein fragment is

[0300] (1) There is a linker fragment 1 between interferon and Pres1

[0301] (2) The linker fragment 1 is between interferon and RBD (SARS-CoV-2)

[0302] (3) The linker sequence between interferon and PADER is linker fragment 1, and the linker fragment between PADER and Pres1 is linker fragment 2

[0303] (4) The linker sequence between interferon and PADER is linker fragment 1, and the linker fragment between PADER and RBD (SARS-CoV-2) is linker fragment 2

[0304] 1.2. Transient transfection to quickly express the target protein:

[0305] (1) Cell recovery: Freestyle 293F cells were cultured at 3×10 7 Cryopreserved in CD OptiCHOTM media (containing 10% DMSO) at a concentration of 10 cells / ml. Removed from liquid nitrogen, quickly thawed in a 37°C water bath, added to a 15ml centrifuge tube containing 10ml OptiCHOTM media and centrifuged at 1,000rpm for 5min. Discarded the supernatant, suspended the cell pellet in 30ml OptiCHOTM media, 37°C, 8% CO2, 135rpm. After 4 days, the cells were expanded and cultured. The concentration during expansion should not exceed 3×10 6 cells / ml.

[0306] (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.

[0307] (3) Two days later, count the transfected cell suspension and estimate the cell density to be 2.5-3.5×10 6 cells / ml, then centrifuge the cell suspension at 1,000 rpm for 5 min and discard the supernatant.

[0308] (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.

[0309] (5) Resuspend the 293F cells in 200 ml Freestyle 293 media.

[0310] (6) Dilute 600 μg of plasmid with 5 ml of Freestyle 293 media and sterilize by filtration using a 0.22 μM filter.

[0311] (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.

[0312] (8) Add the plasmid / PEI mixture to the cell suspension and culture in an incubator at 37°C, 8% CO2, 85rpm, and supplement with 50ug / L LONG™ R3IGF-1.

[0313] (9) After 4 hours, add 200 ml of EX-CELLTM 293 media and 2 mM Glutamine and adjust the rotation speed to 135 rpm to continue culturing.

[0314] (10) After 24 hours, add 3.8 mM VPA, a cell proliferation inhibitor. After 72 hours, add 40 ml of medium D and continue culturing. After 6-8 days (cell survival rate is less than 70%), collect the supernatant for the next purification step.

[0315] 1.3. Collection, purification and electrophoresis verification of fusion protein

[0316] 2. Purify the target protein using Protein A:

[0317] (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.

[0318] (2) Assemble the chromatography column: Take an appropriate amount of Protein A Agarose (calculated as 20 mg human Fc fusion protein per 1 ml of Protein A), mix well and add to the chromatography column. Let it stand at room temperature for about 10 min. After the Protein A and 20% ethanol solution are separated, open the outlet at the bottom to allow the ethanol solution to slowly flow out by gravity.

[0319] (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.

[0320] (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.

[0321] (5) Rinse the column with 10 times the column volume of Binding Buffer to remove impurities and continue washing until no protein is detected in the effluent.

[0322] (6) Use Elution Buffer (0.1 M Glycine, pH 2.7) for elution. Collect the eluate in separate tubes, collecting one tube for every 1 ml, and observe the elution peak using a protein indicator solution (Bio-Rad protein assay). Mix the collection tubes of the elution peak and add an appropriate amount of 1 M Tris, pH 9.0 to neutralize (adjust the pH to 6-8, which should be at least 0.5 different from the isoelectric point of the purified protein).

[0323] (7) Use Zeba desalting centrifugal columns or concentrating centrifugal columns to replace the target protein solution into the required buffer (note to adjust the buffer pH to avoid the isoelectric point of the protein). Use BSA as the standard and determine the protein concentration by SDS-PAGE electrophoresis and NanoDrop2000.

[0324] (8) After elution, rinse the column with 20 times the column volume of distilled water, then rinse the column with 10 times the column volume of 20% ethanol. Finally, the ethanol solution should submerge the gel medium and store at 4°C.

[0325] 3. The protein SDS-PAGE electrophoresis identification diagram is as follows Figure 7 shown.

[0326] Example 3. IFNα-Pres1-Fc and Pres1-Fc can induce stronger immune responses in mice than simple Pres1 antigen.

[0327] Materials: C57BL / 6 male mice (5-8 weeks) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Beijing Kangwei Biotechnology Co., Ltd.; 96-well ELISA assay plates were purchased from Corning Costar; ELISA colorimetric solution was purchased from eBioscience; the microplate reader SPECTRA max PLUS 384 was purchased from Molecular Corporation, USA. The aluminum adjuvant used was purchased from SIGMA.

[0328] method:

[0329] (1) Immunization of mice with Pres1 fusion protein: 80 pmol IFN-Pres1-Fc or 80 pmol Pres1-Fc, Pres1 protein and aluminum adjuvant were mixed and subcutaneously immunized into mice. The serum of mice was collected by orbital bleeding at the designated time points for antibody detection.

[0330] (2) The antibodies produced by IFNα-Pres1-Fc have a broad neutralizing effect on different genotypes of HBV. 5-week-old male C57BL / 6 mice were infected with AAV-HBV 1.3 (HBV genotypes B, C, and D) via the tail vein. 1x10 11 vg virus, and 6 weeks later, mice with continuous and stable expression of HBV antigen were selected for experiments. The selected mice (4 mice / group) were injected intravenously with 200μl / mouse of serum from IFNα-Pres1-Fc immunized mice. After 12 hours, the mouse serum was collected, and the changes of Pres1 antigen in the mice before and after the injection of antiserum were detected by Elisa.

[0331] (3) ELISA detection of anti-Pres1 specific antibodies in serum. Add 50 μl of Pres1 (2 μg / ml) coating solution to the Elisa plate (Corning 9018) at 4°C overnight. Wash once with PBS, 260 μl per well. Block with 5% blocking solution (5% FBS) at 37°C for two hours. Dilute serum samples with PBS (1:10, 1:100, 1:1000, 1:10000), add 50 μl to each well of the blocked Elisa plate and incubate at 37°C for 1 hour. Wash 5 times with PBST, 260 μl each time, add 50 μl of enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS) to each well, and incubate at 37°C for 1 hour. Wash five times with PBST, 260μl each time, add 100μl / well of substrate TMB, incubate at room temperature in the dark, and wait for the substrate to develop color; add 50μl stop solution (2N H2SO4) to each well to stop color development, read the plate with an ELISA reader, OD450-630.

[0332] Results: The immunogenicity of free Pres1 was weak. When IFNα and Fc were added to Pres1 to form IFNα-Pres1-Fc fusion protein, its immunogenicity was greatly improved. Figure 8 (a) As shown. And the antibodies induced by IFNα-Pres1-Fc can produce a wide range of neutralizing effects on different HBV genotypes, such as Figure 8 (b) shown.

[0333] Example 4. IFNα-Pres1-Fc can be used as a preventive vaccine against hepatitis B

[0334] Materials: C57BL / 6 (6-8) week old male mice were purchased from Beijing Weitonglihua Biotechnology Co., Ltd., and HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd. AAV-HBV 1.3 virus was purchased from Guangzhou Paizhen Biotechnology Co., Ltd. Other experimental materials were the same as in Example 3.

[0335] method:

[0336] (1) Mice were subcutaneously immunized with 80 pmol of different forms of Pres1 vaccine, including simple Pres1, Pes1-Fc, and IFNα-Pres1-Fc protein. On day 28 of immunization, mouse sera were collected and mice were infected with 1x10 11vgAAV-HBV 1.3 virus, and then collect mouse serum every week to test anti-Pres1 antibodies, HBsAg, and Pres1 antigen in the serum for four consecutive weeks. The peripheral HBV-DNA level of mice was tested in the third week.

[0337] (2) ELISA detection of Pres1-specific antigen in serum. Antigen coating: Add 50 μl of the coating solution of Pres1 antibody XY007 (4 μg / ml) to the Elisa plate (Corning 9018) per well and coat overnight at 4°C. Wash once with PBS, 260 μl per well. Block with 5% blocking solution (5% FBS) at 37°C for two hours. Dilute the serum sample with PBS (1:10, 1:100), add 50 μl to each well of the blocked Elisa plate, set up two replicate wells for each dilution, and incubate at 37°C for 1 hour. Wash 5 times with PBST, 260 μl each time, add 50 μl of enzyme conjugate (from Kehua HBsAg Detection Kit) to each well, and incubate at 37°C for 1 hour. Wash five times with PBST, 260μl each time, add 100μl / well of substrate TMB, incubate at room temperature in the dark, and wait for the substrate to develop color; add 50μl stop solution (2N H2SO4) to each well to stop color development, read the plate with an ELISA reader, OD450-630.

[0338] Results: The mice in the IFNα-Pres1-Fc immunization group were able to produce high levels of Pres1 antibodies before virus inoculation, and the antibodies continued to maintain a high level during the virus infection. Fig. 9 (a). Compared with the group without protein immunization, IFN-Pres1-Fc vaccine immunization can significantly prevent HBV infection, and the anti-preS1 antibodies produced after immunization can quickly and completely eliminate preS1 antigens in the serum Fig. 9 (b) In addition, most of the mice infected with the virus in the IFN-Pres1-Fc immunization group showed peripheral HBsAg negative Fig. 9 (c, d). The above experimental results show that IFN-Pres1-Fc as a vaccine can effectively prevent HBV infection. Fig. 9 shown.

[0339] Example 5. IFNα-Pres1-Fc as a therapeutic vaccine for chronic influenza B infection

[0340] Materials: C57BL / 6 male mice (4 weeks old) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. AAV-HBV 1.3 was purchased from Guangzhou Paizhen Biotechnology Co., Ltd. HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd. Other experimental materials were the same as in Example 4.

[0341] method:

[0342] (1) Screening of HBV Carrier Mice: 4-week-old HBV C57BL / 6 mice were injected with 1x10 11 vg AAV-HBV 1.3 virus, HBV antigen HBsAg was detected in 1-6 weeks, and mice with stable HBsAg expression were screened out and used as HBV Carrier mice for experiments.

[0343] (2) The selected mice were injected subcutaneously with 80 pmol of different forms of Pres1 protein, once every two weeks for a total of three immunizations. Mouse serum was collected on day 14 of immunization and then once a week. The levels of anti-Pres1 antibodies, HBsAg, and Pres1 antigen in the mouse serum were tested by ELISA. The HBV-DNA content in the mouse peripheral blood was tested after the last blood draw.

[0344] Results: We detected the changes in serum preS1 antigen, serum Pres1 antibody and HBsAg in Carrier mice after immunization with IFNα-Pres1-Fc vaccine. The results showed that after IFNα-Pres1-Fc vaccine immunization, mice produced high levels of anti-Pres1 antibodies. Fig.10 (a), and the preS1 antigen in the serum can be completely cleared, such as Fig.10 (b) As shown, HBsAg in serum also decreased to a certain extent as shown in Figure 10(c), while the untreated control group and the group immunized with Pres1 vaccine alone had no therapeutic effect. Fig.10 shown.

[0345] Example 6. T cell helper epitopes enhance the antibody response to IFNα-Pres1-Fc vaccine

[0346] Materials: Same as Example 3

[0347] method:

[0348] (1) Immunize mice with Pres1 fusion protein. Immunize mice subcutaneously with 80 pmol of IFN-Pan-Pres1-Fc containing the Pan epitope or 80 pmol of IFN-Pan-Pres1-Fc, Pres1-Fc, and Pres1 protein. Collect serum from mice by orbital bleeding at designated time points for antibody detection.

[0349] (2) ELISA was used to detect anti-Pres1 specific antibodies in serum, as in Example 3.

[0350] Results: Compared with fusion protein vaccines such as IFN-preS1-Fc, IFN-Pan-preS1-Fc can significantly enhance the immunogenicity of antigen molecules and induce the production of broad-spectrum neutralizing antibodies. C57 / BL6 (n=8 / group) mice were subcutaneously immunized with hepatitis B Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins without aluminum adjuvant, and the level of Pres1-specific antibodies in serum was detected by Elisa at the designated time.

[0351] Example 7. IFNα-Pan-Pres1-Fc as a therapeutic vaccine for chronic influenza B infection

[0352] Materials: C57BL / 6 male mice (4 weeks old) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. AAV-HBV 1.3 was purchased from Guangzhou Paizhen Biotechnology Co., Ltd. HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd. Other experimental materials were the same as in Example 4.

[0353] method:

[0354] (1) Screening of HBV Carrier Mice: 4-week-old HBV C57BL / 6 mice were injected with 1x10 11 vg AAV-HBV 1.3 virus, HBV antigen HBsAg was detected in 1-6 weeks, and mice with stable HBsAg expression were screened out and used as HBV Carrier mice for experiments.

[0355] (2) The selected mice were injected subcutaneously with 80 pmol of different forms of Pres1 protein, once every two weeks for a total of three immunizations. Mouse serum was collected on day 14 of immunization, and then collected once a week. The levels of anti-Pres1 antibodies, HBsAG, and Pres1 antigen in the mouse serum were tested by ELISA. The HBV-DNA content in the mouse peripheral blood was tested after the last blood draw.

[0356] Results: We detected the changes in serum preS1 antigen, serum Pres1 antibody and HBsAg in Carrier mice after immunization with IFN-Pan-Pres1-Fc vaccine. The results showed that after immunization with IFN-Pan-Pres1-Fc vaccine, mice produced high levels of anti-Pres1 antibodies such as Fig.12 (a) As shown. And accompanied by the complete removal of preS1 antigen in serum Fig.12(b) As shown, the HBsAg in the serum also decreased to a certain extent12(c), while the untreated control group and the group immunized with Pres1 vaccine alone had no therapeutic effect. In addition, the HBV DNA also decreased significantly in the IFNα-Pan-Pres1-Fc immunization group. Fig.12 (d) as shown.

[0357] Example 8. IFNα-Pan-Pres1-Fc combined with HBsAg commercial vaccine breaks the immune tolerance caused by HBsAg and induces HBsAg-HBsAb serological conversion.

[0358] 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.

[0359] method:

[0360] (1) Screening of HBV Carrier Mice: 4-week-old HBV C57BL / 6 mice were injected with 1x10 11 vg AAV-HBV 1.3 virus, HBV antigen HBsAg was detected in 1-6 weeks, and mice with stable HBsAg expression were screened out and used as HBV Carrier mice for experiments.

[0361] (2) The HBV Carrier mice screened were immunized with 80 pmol IFNα-pan-Pres1-Fc and 2 μg of commercial HBsAg vaccine twice, with an interval of 14 days between each immunization. Mouse serum was collected 14 days after the first immunization, and then weekly, and the changes in anti-Pres1, Pres1, anti-HBsAg, and HBsAg in the serum were detected. The level of HBV-DNA in the serum was detected at the last mouse serum collection.

[0362] Results: We found that the combination of IFNα-Pan-Pres1-Fc and commercial HBsAg as a strategy for treating chronic hepatitis B can ultimately break HBsAg tolerance. The immune response generated in HBV-tolerant mice can completely eliminate preS1 antigen in the serum Fig.13(a), and there are high concentrations of Pres1 antibodies in the serum13 (c). Excitingly, the IFN-Pan-Pres1-Fc vaccine effectively cleared HBsAg in the serum and induced some serological HBsAb conversion Fig.13 (b) and 4(d), which are clinically considered to be the key indicators 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. Fig.13 (e) Based on the above results, we invented a vaccine strategy combining IFNα-Pan-Pres1-Fc and commercial HBsAg vaccine for the treatment of chronic hepatitis B

[0363] Example 9. IFNα-RBD(SARS-CoV2)-Fc can induce stronger antibody response than free SARS-Cov2 RBD protein

[0364] Materials: Balb / c male and female mice (6-8 weeks) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., and the SARS-CoV-2 RBD protein used 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 was purchased from Promega.

[0365] Other experimental materials are the same as in Example 3.

[0366] method:

[0367] (1) Immunization of mice with IFNα-RBD (SARS-Cov-2)-Fc fusion protein: 10 μg IFNα-RBD-Fc, RBD-Fc or 10 μg RBD protein was mixed with aluminum adjuvant and subcutaneously immunized into mice. The serum of mice was collected by orbital bleeding on day 28 of immunization for detection of coronavirus-specific antibodies.

[0368] (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) at 4°C overnight. 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 to each well of the blocked Elisa plate and incubate at 37°C for 1 hour. Wash 5 times with PBST, 260 μl each time, add 100 μl of enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000diluted by PBS) to each well, and incubate at 37°C for 1 hour. Wash five times with PBST, 260μl each time, add substrate TMB 100μl / well, incubate at room temperature in the dark for 15 minutes, and wait for the substrate to develop color. Add 50μl stop solution (2N H2SO4) to each well to stop the color development, read the plate with an ELISA reader, OD450-630. The titer calculation method is to select the maximum dilution factor with a positive result, multiply the OD value / Cutoff value (0.1) corresponding to the dilution factor by (X) the dilution factor, and the resulting value is the antibody titer corresponding to the serum.

[0369] (3) SARS-CoV-2 S protein pseudovirus in vitro neutralization experiment. Antiserum was diluted 1:3 and added to a 96-well plate. 50ul of pseudovirus particles with luciferase spike protein were added to the wells. The virus-antibody mixture was placed at 37ºC 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 37ºC cell culture incubator and the activity of luciferase was detected after 48 hours.

[0370] Results: The immunogenicity of free coronavirus RBD was weak. When IFNα and Fc were added to the coronavirus RBD polypeptide protein region to form IFNα-RBD-Fc fusion protein, its immunogenicity was greatly improved. Fig.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. Fig.15 shown.

[0371] 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.

[0372] Materials: Balb / c male and female mice (6-8 weeks) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and the SARS-CoV-2 original strain RBD protein was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd. The SARS-CoV-2 South African mutant strain RBD protein was purchased from Beijing Sino Biological Technology Co., Ltd.

[0373] Other experimental materials are the same as in Example 3.

[0374] method:

[0375] (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.

[0376] (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. The serum of mice was collected by orbital bleeding on day 14 of immunization for detection of coronavirus-specific antibodies.

[0377] (3) Analysis of antibody response by ELISA method is the same as in Example 9.

[0378] Results: SDS-PAGE results showed the correct band size of IFNα-Pan-RBD (original strain of SARS-CoV-2)-Fc, indicating that the mutant coronavirus IFNα-RBD (original strain of SARS-CoV-2)-Fc vaccine protein was successfully constructed, expressed, and purified ( Fig.16 a), SDS-PAGE results showed the correct band size of IFNα-Pan-RBD (SARS-CoV-2 South African mutant)-Fc, indicating that the mutant coronavirus IFNα-RBD (SARS-CoV-2 South African mutant)-Fc vaccine protein was successfully constructed, expressed, and purified ( Fig.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 strain)-Fc could bind to the RBD protein of the original strain coronavirus, and there was no significant difference in the ability of the antibodies induced by the two to bind to the RBD of the original strain ( Fig.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, and their binding abilities are comparable ( Fig.16 d).

[0379] Example 11.

[0380] 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.; 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 this laboratory, and other experimental materials were the same as in Example 3.

[0381] method:

[0382] (1) The fusion protein design, plasmid construction and protein purification methods are the same as those in Examples 1 and 2.

[0383] (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 proteins were 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. 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.

[0384] (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) at 4°C overnight. 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 to each well of the blocked Elisa plate and incubate at 37°C for 1 hour. Wash 5 times with PBST, 260 μl each time, add 100 μl of enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000diluted by PBS) to each well, and incubate at 37°C for 1 hour. Wash five times with PBST, 260μl each time, add substrate TMB 100μl / well, incubate at room temperature in the dark for 15 minutes, and wait for the substrate to develop color. Add 50μl stop solution (2N H2SO4) to each well to stop the color development, read the plate with an ELISA reader, OD450-630. The titer calculation method is to select the maximum dilution factor with a positive result, multiply the OD value / Cutoff value (0.1) corresponding to the dilution factor by (X) the dilution factor, and the resulting value is the antibody titer corresponding to the serum.

[0385] result:

[0386] like Fig.17 As shown, 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.

[0387] like Fig.18 As shown, the addition of Pan (Pan DR-binding epitope) CD4 T cell helper epitope can enhance the immunity of Mouse IFNα-RBD-Fc and Human IFNα-RBD-Fc. The experimental results show that whether it is on the 7th day, 14th day, or 28th day after vaccine protein immunization, the addition of Pan epitope can induce higher RBD-specific antibody production in Mouse IFNα-Pan-RBD-Fc compared with Mouse IFNα-RBD-Fc, and Human IFNα-Pan-RBD-Fc compared with Human IFNα-RBD-Fc.

[0388] Example 12

[0389] 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 this laboratory. Other experimental materials were the same as in Example 3.

[0390] method:

[0391] (1) Immunization of mice with Human IFNα-RBD-Fc and 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 as the vaccine sample containing aluminum adjuvant. Another group of 10 μg of Human IFNα-RBD-Fc or Human IFNα-Pan-RBD-Fc protein was diluted with PBS as the vaccine sample 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 level of RBD-specific antibodies in the mouse sera was detected by ELISA.

[0392] (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) at 4°C overnight. 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 to each well of the blocked Elisa plate and incubate at 37°C for 1 hour. Wash 5 times with PBST, 260 μl each time, add 100 μl of enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000diluted by PBS) to each well, and incubate at 37°C for 1 hour. Wash five times with PBST, 260μl each time, add substrate TMB 100μl / well, incubate at room temperature in the dark for 15 minutes, and wait for the substrate to develop color. Add 50μl stop solution (2N H2SO4) to each well to stop the color development, read the plate with an ELISA reader, OD450-630. The titer calculation method is to select the maximum dilution factor with a positive result, multiply the OD value / Cutoff value (0.1) corresponding to the dilution factor by (X) the dilution factor, and the resulting value is the antibody titer corresponding to the serum.

[0393] result:

[0394] like Fig.19 As shown in the results, the application of aluminum adjuvant can enhance the immunogenicity of Human IFNα-RBD-Fc and Human IFNα-Pan-RBD-Fc proteins. Although the non-adjuvanted Human IFNα-RBD-Fc and Human IFNα-Pan-RBD-Fc vaccines can produce high-titer antibody responses, the aluminum adjuvant-assisted Human IFNα-RBD-Fc and Human IFNα-Pan-RBD-Fc proteins can further improve the RBD-specific antibody response level compared with the non-adjuvant-assisted group on the 7th, 14th and 28th days after vaccination.

[0395] Example 13.

[0396] Material:

[0397] The experimental animals were purchased from Beijing Weitonglihua Experimental Animal Co., Ltd., and the animals used were 6-8 week old C57BL / 6 mice; animal 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 by 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 assay plates were purchased from Corning Costar; ELISA colorimetric solution was purchased from eBioscience; the microplate reader SPECTRA max PLUS 384 was purchased from Molecular Corporation of the United States; and the tissue homogenizer was purchased from Beijing Haonuosi Technology Co., Ltd.

[0398] method:

[0399] 6-8 week old mice were divided into 5 groups, 10 mice in each group, and immunized with 10 μg of IFNα-pan-RBD-Fc or the same molar number of RBD, RBD-Fc, IFNα-RBD-Fc protein by intranasal immunization, with an intranasal dose of 10 μL per mouse. Mice were immunized twice on days 0 and 14. Mouse sera were collected on days 7, 14, 21, 28, 35, and 42 after immunization, and the ELISA method was used to detect the content of SARS-CoV-2 RBD-specific antibodies in the sera of each group; the sera taken on day 28 were used for in vitro neutralization experiments of SARS-CoV-2 pseudoviruses.

[0400] result:

[0401] like Fig. 20 As shown, two nasal drops 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 nasal drops of IFNα 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.

[0402] Example 14.

[0403] Material:

[0404] Same as Example 10

[0405] method:

[0406] 6-8 week old mice were divided into 4 groups, 5 in each group, and immunized with 10 μg of IFNα-pan-RBD-Fc or the same molar number of RBD, RBD-Fc, IFNα-RBD-Fc protein by intranasal immunization, with a dose of 10 μL per mouse. The mice were immunized twice on days 0 and 14. The nasal mucosal supernatant and lung lavage fluid of the mice were collected on day 28 after immunization, and the SARS-CoV-2 RBD-specific antibody content in the serum of each group was detected by ELISA, and the coronavirus pseudovirus neutralization experiment of the serum and nasal mucosal supernatant was measured by SARS-CoV-2 pseudovirus neutralization experiment.

[0407] Acquisition of nasal mucosal supernatant and alveolar lavage fluid of mice in immune experimental animals: After the mice were dormant and killed, the nasal mucosa of the mice was taken and broken with a tissue homogenizer. The homogenized liquid was centrifuged at 13000rpm for 10 minutes, and the supernatant was the nasal mucosal supernatant (NMDS). For the mouse lungs, use a 1ml syringe to draw about 0.8ml of HBSS+100μMEDTA, inject it into the tracheal cannula, and gently blow and aspirate three times, then aspirate the liquid and collect it in a centrifuge tube. Repeat this step three times, and finally about 2ml of lung lavage fluid can be obtained. Centrifuge the mouse lung lavage fluid at 500g for 5 minutes, the supernatant is the mouse lung lavage fluid (BALF), and the precipitate is the lymphocytes in the mouse lungs, which can be further analyzed.

[0408] result:

[0409] like Fig.21 As shown, compared with RBD and RBD-Fc proteins, two nasal drops of IFNα-pan-RBD-Fc protein can induce strong local IgG antibody response and IgA mucosal immunity in the nasal mucosa. The intensity of the IFNα-pan-RBD-Fc protein reaction is stronger than that of the RBD and RBD-Fc groups. The results of the pseudovirus neutralization experiment showed that the IFNα-pan-RBD-Fc protein immunization group can induce higher titers of neutralizing antibodies in the nasal mucosa.

[0410] like Fig. 22 As shown, two intranasal immunizations of C57BL / 6 mice with IFNα-pan-RBD-Fc fusion protein also induced strong secretion of IgG and IgA antibodies in local lung tissue. The results of pseudovirus neutralization experiments showed that IFNα-pan-RBD-Fc induced higher titers of neutralizing antibodies than RBD and RBD-Fc.

[0411] Example 15.

[0412] Her2 belongs to the type I transmembrane growth factor receptor HER family, which consists of an extracellular ligand binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. Once the ligand binds to the extracellular domain, the HER protein will dimerize and trans-phosphorylate its intracellular domain. The phosphorylated tyrosine residues can bind to a variety of intracellular signaling molecules, activate downstream signaling pathways, and regulate gene transcription. The regulated genes are mostly related to processes such as cell proliferation, survival, differentiation, angiogenesis, invasion, and metastasis. The extracellular segment of the Her2 protein is large, with more than 600 amino acids, and can be divided into four domains, namely domains I, II, III, and IV. The currently approved Trastuzumab mainly binds to domain IV, Pertuzumab mainly binds to domain II, and the peptide vaccine E75, which is currently undergoing clinical trials, targets domain III. This shows that there are some important sites in different domains 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.

[0413] Materials and methods:

[0414] Material:

[0415] BALB / c female mice (6-8 weeks) were purchased from Beijing Weitonglihua Biotechnology Co., Ltd.; TUBO cells were from TCGA; other materials were the same as in Example 3.

[0416] method:

[0417] (1) Fusion protein design, plasmid construction and protein purification methods are shown in Examples 1 and 2.

[0418] First, expression plasmids were constructed for the extracellular domains III and IV of mouse Her2 (respectively: IFNα-3-Fc, IFNα-pan-3-Fc, IFNα-pan-4-Fc and IFNα-4-Fc), and the related proteins were expressed and purified in the human 293F cell line. The size and purity of the proteins were identified by SDS-PAGE and Coomassie Brilliant Blue staining.

[0419] (2) Analysis of direct antitumor activity of IFNα-3-Fc and IFNα-pan-3-Fc

[0420] 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 tested. TUBO breast cancer model mice were constructed, 5*10 5TUBO cells were subcutaneously inoculated into BALB / C mice, and the tumor size was 50-80 mm 3 The treatment was given once a week for a total of 3 times. The dosage of IFNα-3-Fc was 10ug / mouse, and other drugs were given in equal moles. CpG was used as an adjuvant. The tumor size was measured and the tumor growth curve was drawn.

[0421] (3) Analysis of the effect of IFNα and Pan on the immunogenicity of Her2 vaccine

[0422] BALB / C female mice aged 6-8 weeks were subcutaneously inoculated with HER2 domain V fusion protein vaccines 4-Fc, IFNα-4-Fc and IFNα-pan-4-Fc without adjuvant, once a week for a total of 3 times. The immunization dose was IFNα-4-Fc 10ug / mouse, and other proteins were inoculated in equal moles. Venous blood was collected on days 14 and 21 after immunization, and the antibody level of Her2-specific IgG was detected by ELISA.

[0423] result:

[0424] (1) If Fig.23 As shown, the size of Her2 fusion protein is basically in line with expectations, and the purity meets the experimental requirements. IFNα-3-Fc (62.6kDa), IFNα-pan-3-Fc (63.9kDa), IFNα-pan-4-Fc (74.9kDa) and IFNα-4-Fc (73.6kDa). Under non-deformation conditions, the protein is in a dimer state, which is consistent with the characteristics of automatic dimerization of the Fc fragment.

[0425] (2) If Fig.24 As shown in the figure, compared with the control group, intratumoral injection of 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 activity of IFNα in the protein vaccine is good, and 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.

[0426] (3) If Fig.25As shown in the figure, 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 can all induce significant Her2-specific IgG antibody responses; compared with 4-Fc, the antibody titers induced by IFNα-4-Fc and IFNα-pan-4-Fc tend to increase. Moreover, at 21 days after immunization, the antibody titer induced by IFNα-pan-4-Fc was significantly higher than that of the 4-Fc group. This indicates that the addition of IFNα and pan helps to increase the immunogenicity of 4-Fc and induce a stronger antigen-specific antibody response. Therefore, IFN-Pan-HER2-Fc and IFN-Pan-HER2-Fc are potential effective tumor vaccines against Her2-positive tumors.

[0427] Example 16.

[0428] 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 this laboratory; H1N1 (A / PR8) influenza virus used to infect mice was produced in this laboratory; other experimental materials were the same as in Example 3.

[0429] method:

[0430] (1) The design, plasmid construction and protein purification of IFNα-HA1-Fc protein were the same as those in Examples 1 and 2.

[0431] (2) Immunize mice with HA1 and IFNα-HA1-Fc proteins. 10 μg IFNα-HA1-Fc or the same molar amount of HA1 protein was mixed with 20 μg aluminum adjuvant overnight and then inoculated into mice by intramuscular immunization. A booster immunization was performed 14 days after the initial vaccination. Mouse serum was collected on the 28th day after immunization, and the level of HA1-specific antibodies in the mouse serum was detected by ELISA.

[0432] (3) Detection of serum HA1 antibodies. Antigen coating: Add HA1 (2μg / ml) coating solution to the Elisa plate (Corning 9018) at 100ul per well and coat overnight at 4°C. Wash once with PBS, 260μl per well. Block with 100μl 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 100μl to each well of the blocked Elisa plate and incubate at 37°C for 1 hour. Wash 5 times with PBST, 260ul each time, add 100μl of enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS) to each well, and incubate at 37°C for 1 hour. Wash five times with PBST, 260μl each time, add substrate TMB 100μl / well, incubate at room temperature in the dark for 15 minutes, and wait for the substrate to develop color. Add 50μl stop solution (2N H2SO4) to each well to stop the color development, read the plate with an ELISA reader, OD450-630. The titer calculation method is to select the maximum dilution factor with a positive result, multiply the OD value / Cutoff value (0.1) corresponding to the dilution factor by (X) the dilution factor, and the resulting value is the antibody titer corresponding to the serum.

[0433] (4) 42 days after the mice were immunized, they were anesthetized and infected with 1000 PFU A / PR8 influenza virus via nasal drops. Starting from the third day after infection, the mice were observed and weighed every two days.

[0434] result:

[0435] like Fig.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, and the results showed that a single band was present at the target band size position. Fig. 27 As shown, IFNα-HA1-Fc can induce higher titers of HA1-specific antibodies compared with HA1 protein ( Fig. 27 a), indicating that the vaccine platform can enhance the immunogenicity of HA1 protein. The weight of mice will change significantly after the virus attack, 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 ( Fig. 27 b).

[0436] Embodiment 17

[0437] Materials and methods:

[0438] 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 are the same as those shown in Examples 1 and 2.

[0439] result:

[0440] like Fig.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 band was in the correct position.

[0441] Embodiment 18

[0442] IFNa-Pan-EBV gp350-Fc induces a stronger humoral immune response than free EBV-gp350 protein

[0443] 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 for immunization was purchased from Taizhou Baiying Biotechnology Co., Ltd., and IFNα-Pan-EBV gp350-Fc was produced in this laboratory; other experimental materials were the same as in Example 3.

[0444] method:

[0445] (1) Immunization of mice with EBV gp350 and IFNα-Pan-EBV gp350-Fc proteins. 10 μg IFNα-Pan-EBVgp350-Fc or the same molar amount of EBV-gp350 protein was mixed with 20 μg aluminum adjuvant overnight, and then the mice were inoculated by intramuscular immunization. A booster immunization was performed 14 days after the initial vaccination. The mouse sera were collected on the 14th and 28th days after the initial immunization, and the EBV-gp350-specific antibody level in the mouse sera was detected by ELISA.

[0446] (2) Detection of serum EBV-gp350 antibodies. Antigen coating: Add 100 μl of EBV-gp350 (1 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 ul 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, 260μl each time, add 100μl enzyme-conjugated anti-mouse IgG-HRP 1:5000diluted by PBS to each well, and incubate at 37°C for 0.5 hours. Wash five times with PBST, 260μl each time, add 100μl / well of substrate TMB, incubate at room temperature in the dark for 15 minutes, and wait for the substrate to develop color. Add 50μl stop solution (2N H2SO4) to each well to stop the color development, read the plate with an enzyme reader, OD450-630. The titer calculation method is to select the maximum dilution factor with a positive result, and multiply (X) the dilution factor by the OD value / Cutoff value (0.1) corresponding to the dilution factor, and the resulting value is the antibody titer corresponding to the serum.

[0447] Results: Fig.29 As shown, 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 double immunization (boost), which fully demonstrates that this vaccine platform can significantly compete for the immunogenicity of EBV-gp350.

[0448] Embodiment 19

[0449] IFNa-Pan-HSV-2 gD-Fc can induce a stronger humoral immune response than free HSV-2 gD protein.

[0450] 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 for immunization was purchased from Taizhou Baiying Biotechnology Co., Ltd., and IFNa-Pan-HSV-2 gD-Fc was produced in this laboratory; other experimental materials were the same as in Example 3.

[0451] method:

[0452] (1) Mice were immunized with HSV-2 gD and 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 into the mice by intramuscular immunization. A booster immunization was performed 14 days after the initial immunization. Mouse sera were collected on the 14th and 28th days after the initial immunization, and the EBV-gp350-specific antibody level in the mouse sera was detected by ELISA.

[0453] (2) Detection of serum HSV-2 gD antibodies. Antigen coating: Add 100 μl of HSV-2 gD (1 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 5 times with PBST, 260μl each time, add 100μl enzyme-conjugated anti-mouse IgG-HRP 1:5000diluted by PBS to each well, and incubate at 37°C for 0.5 hours. Wash five times with PBST, 260μl each time, add 100μl / well of substrate TMB, incubate at room temperature in the dark for 15 minutes, and wait for the substrate to develop color. Add 50μl stop solution (2N H2SO4) to each well to stop the color development, read the plate with an enzyme reader, OD450-630. The titer calculation method is to select the maximum dilution factor with a positive result, and multiply (X) the dilution factor by the OD value / Cutoff value (0.1) corresponding to the dilution factor, and the resulting value is the antibody titer corresponding to the serum.

[0454] Results: Fig.30 As shown, 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 double immunization (boost), which fully demonstrates that this vaccine platform can significantly enhance the immunogenicity of HSV-2 gD.

[0455] 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.

[0456] 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 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 this laboratory; other experimental materials were the same as in Example 3.

[0457] method:

[0458] (1) Mice were immunized with VZV gE and IFNa-Pan-VZV gE-Fc proteins. 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 the mice were inoculated 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.

[0459] (2) Detection of total serum VSV gE antibodies. Antigen coating: Add 100 μl of VSV gE (1 μg / ml) coating solution to the ELISA plate (Corning 9018) at 4°C overnight. 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 to each well of the blocked ELISA plate and incubate at 37°C for 1 hour. Wash 5 times with PBST, 260 μl each time, add 100 μl of enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000diluted by PBS) to each well, and incubate at 37°C for 0.5 hour. Wash five times with PBST, 260ul each time, add 100μl / well of substrate TMB, incubate at room temperature in the dark for 15 minutes, and wait for the substrate to develop color. Add 50μl of stop solution (2N H2SO4) to each well to stop the color development, read the plate with an ELISA reader, OD450-630. The titer calculation method is to select the maximum dilution factor with a positive result, multiply the OD value / Cutoff value (0.1) corresponding to the dilution factor by (X) the dilution factor, and the resulting value is the antibody titer corresponding to the serum.

[0460] (3) Detection of serum VSV gE-specific antibody subtypes. Antigen coating: Add 100 μl of VSV gE (1 μg / ml) coating solution to the ELISA plate (Corning 9018) at 4°C overnight. 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 serum samples 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 5 times with PBST, 260 μl each time, 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 five times with PBST, 260ul each time, add 100ul / well of substrate TMB, incubate at room temperature in the dark for 15 minutes, and wait for the substrate to develop color. Add 50μl of stop solution (2N H2SO4) to each well to stop the color development, read the plate with an ELISA reader, OD450-630. The titer calculation method is to select the maximum dilution factor with a positive result, multiply (X) the dilution factor by the OD value / Cutoff value (0.1) corresponding to the dilution factor, and the resulting value is the antibody titer corresponding to the serum.

[0461] 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). Fig.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 ( Fig.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 demonstrated that IFNa-Pan-VZV gE-Fc can not only produce a stronger humoral immune response than free VZV-gE protein, but also induce a Th1 / Th2 balanced immune response.

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Claims

1. A vaccine comprising a fusion protein with interferon, a target antigen, and an immunoglobulin Fc region as structural units, wherein the interferon is the first structural unit, wherein the first structural unit is IFN-α, and wherein the immunoglobulin Fc region is the second structural unit, which is selected from the Fc region of IgG immunoglobulin, in, The target antigen is the third structural unit, wherein the target antigen is the gE antigen of herpes zoster virus VZV, the gD antigen of herpes simplex virus HSV-2, the full-length antigen or S1 region antigen of SARS-COV-2 virus S protein, or HER2 antigen, or The target antigen is a mutated viral antigen different from the wild type, the virus is SARS-COV-2, and the mutated viral antigen different from the wild type is selected from a SARS-COV-2 viral antigen mutant with a sequence shown in any one of SEQ ID NO.79, SEQ ID NO.80, SEQ ID NO.81, SEQ ID NO.82, SEQ ID NO.83 or SEQ ID NO.

84.

2. The vaccine according to claim 1, which has any of the following characteristics: 1) The target antigen is the gE antigen of herpes zoster virus VZV; 2) The first structural unit is derived from human or mouse; 3) The first structural unit is selected from mouse IFN-α4, human IFN-α2, and a human IFN-α2 mutant that binds to human and mouse IFN receptors; 4) The first structural unit is selected from the interferon represented by the amino acid sequence of SEQ ID NO.1, SEQ ID NO.21, and SEQ ID NO.22; 5) The second structural unit is selected from the Fc region of the constant region of IgG1; 6) The second structural unit is selected from the Fc region shown in SEQ ID NO.2, SEQ ID NO.23, and SEQ ID NO.24; 7) The target antigen is the SARS-COV-2 antigen shown in SEQ ID NO.76 or SEQ ID NO.77; 8) The vaccine is a targeted vaccine; 9) The fusion protein contains one or more Th cell helper epitopes and / or linker fragments, or 10) The structural units are connected in the fusion protein in the order of IFN-α-target antigen-Fc region or Fc region-target antigen-IFN-α.

3. The vaccine according to claim 1 or 2, which has any of the following characteristics: 1) The third structural unit is a viral antigen, which is fused with an auxiliary polypeptide epitope that enhances B cell and T cell responses and is located at the N-terminus or C-terminus of the antigen epitope. 2) The third structural unit is a viral antigen, and the viral antigen is fused with a Pan HLA DR-binding epitope, 3) The third structural unit is a viral antigen, and the viral antigen is fused with the epitope shown in the amino acid sequence of SEQ ID NO.3; 4) The structural units are connected by connecting segments, and the connecting segments are flexible polypeptide sequences, 5) The structural units are connected via a connecting fragment, the sequence of the connecting fragment is the amino acid sequence shown in SEQ ID NO.4 or SEQ ID NO.25, 6) Each polypeptide sequence composed of the structural unit contains a signal peptide at the N-terminus that can promote protein secretion. 7) Each polypeptide sequence composed of the structural unit contains a signal peptide represented by the amino acid sequence of SEQ ID NO.5 at the N-terminus, 8) The vaccine is produced by a eukaryotic expression system, or 9) The vaccine is produced by the eukaryotic expression system 293F or CHO cells.

4. The vaccine according to claim 1 or 2, wherein the target antigen is selected from the Her2 antigen shown in SEQ ID NO. 85, 86, 87, 88, 89, and 90.

5. The vaccine of claim 1 or 2, wherein the fusion protein is a homodimer or heterodimer fusion protein comprising a first polypeptide chain and a second polypeptide chain.

6. The vaccine according to claim 5, which has any of the following characteristics: 1) The fusion protein comprises one or more Th cell helper epitopes and / or connecting fragments in the first polypeptide chain and / or the second polypeptide chain of the homodimer or heterodimer; 2) the first polypeptide chain and the second polypeptide chain of the homodimer are completely identical; 3) the first polypeptide chain and the second polypeptide chain of the homodimer sequentially comprise IFN-α, target antigen and immunoglobulin Fc region from N-terminus to C-terminus, and form a homodimer; 4) the homodimer comprises a Th cell helper epitope; 5) the first polypeptide chain and the second polypeptide chain of the heterodimer are different; or 6) The heterodimer comprises a Th cell helper epitope.

7. The vaccine according to claim 5, wherein The first polypeptide chain and the second polypeptide chain of the homologous dimer comprise IFN-α, gE antigen of VZV and immunoglobulin Fc region from N-terminus to C-terminus.

8. A nucleic acid molecule encoding the fusion protein in the vaccine according to any one of claims 1 to 7.

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

10. A host cell comprising the nucleic acid molecule of claim 8 or the expression vector of claim 9. The host cell according to claim 10 , which is a eukaryotic cell.

12. Use of the fusion protein defined in any one of claims 1 to 7 in the preparation of vaccines, compositions, kits, recombinant microorganisms or cell lines.

13. The use according to claim 12, wherein the composition is a pharmaceutical composition or an immunogenic composition.

14. The use according to any one of claims 12-13, which has any of the following features: 1) The vaccine, the composition or the kit further comprises an adjuvant; or 2) The vaccine, the composition or the kit comprises aluminum adjuvant (Alum), Toll-like receptor 4 activator ligand MPLA, Toll-like receptor 9 ligand, oligodeoxynucleotide (CpG-ODN), MF59 and Freund's adjuvant.

15. Use of the fusion protein defined in any one of claims 1 to 7 for the preparation of a preventive or therapeutic vaccine against VZV, HSV-2 or SARS-COV-2.

16. The use according to claim 15, having any of the following features: 1) The vaccine is used in combination with another preventive or therapeutic therapy; 2) Combination use of the vaccine with antiviral drugs and other treatments; 3) The vaccine is used as a component of a vaccine to form a multivalent combination vaccine with other viruses, pathogens or tumor vaccines, 4) The vaccine is a SARS-COV-2 vaccine, which is used in combination with an influenza vaccine or other vaccines to form a multivalent vaccine. 5) The vaccine is administered in a sequential or simultaneous manner with an adenovirus vaccine, mRNA vaccine, inactivated vaccine or DNA vaccine for the same pathogen or tumor; 6) The vaccine is administered via intramuscular, intravenous, transdermal, subcutaneous or nasal immunization routes; 7) The vaccine also includes an adjuvant; 8) The vaccine comprises aluminum adjuvant (Alum), Toll-like receptor 4 activator ligand MPLA, Toll-like receptor 9 ligand, oligodeoxynucleotide (CpG-ODN), MF59 and Freund's adjuvant, or 9) The vaccine is a SARS-COV-2 fusion protein vaccine, which is immunized with adenovirus vaccine, mRNA vaccine, inactivated vaccine or DNA vaccine of SARS-COV-2 in a sequential or simultaneous immunization program, wherein the sequential immunization order is: 1) first immunize with SARS-COV-2 fusion protein vaccine, then immunize with adenovirus vaccine, mRNA vaccine, inactivated vaccine or DNA vaccine of SARS-COV-2; 2) first immunize with adenovirus vaccine, mRNA vaccine, inactivated vaccine or DNA vaccine of SARS-COV-2, then immunize with SARS-COV-2 fusion protein vaccine; 3) immunize with SARS-COV-2 fusion protein vaccine and adenovirus vaccine, mRNA vaccine, inactivated vaccine or DNA vaccine of SARS-COV-2 simultaneously.

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