CN115137812A

CN115137812A - Construction and application of fusion protein vaccine platform

Info

Publication number: CN115137812A
Application number: CN202111680865.3A
Authority: CN
Inventors: 彭华; 孙士玉; 其他发明人请求不公开姓名
Original assignee: Institute of Biophysics of CAS
Current assignee: Institute of Biophysics of CAS
Priority date: 2021-03-31
Filing date: 2021-12-31
Publication date: 2022-10-04
Anticipated expiration: 2041-12-31
Also published as: CN115137812B; CN120022357A; CN120459288A

Abstract

The invention relates to construction and application of a fusion protein vaccine platform. The invention provides a vaccine which comprises a fusion protein of interferon-target antigen-immunoglobulin Fc region (or antibody) and Th cell helper epitope. The invention also relates to the use of a fusion protein comprising an interferon-target antigen-immunoglobulin Fc (or antibody) region and a Th cell helper epitope for the preparation of a prophylactic or therapeutic composition. The vaccine of the invention can be produced by a eukaryotic cell expression system, can be used for preparing wild type and various mutant antigen vaccines, and can cause strong immune response of organisms by vaccination through subcutaneous/muscle or nasal cavity and other immunization ways. The vaccine of the present invention may be used as a prophylactic or therapeutic vaccine.

Description

Construction and application of fusion protein vaccine platform

Technical Field

The invention belongs to the technical field of genetic engineering and biomedicine, and particularly relates to a vaccine, such as a fusion protein vaccine which contains interferon-target antigen-immunoglobulin Fc region (antibody) as a main framework. The vaccine can be used as a vaccine platform for preventing Hepatitis B Virus (HBV) infection, HPV, EBV, HIV, SARA-CoV-2, influenza virus infection and HPV and EBV related tumor occurrence, treating Chronic Hepatitis B (CHB) infection and treating HBV, HPV and EBV related tumor.

Background

About 2.57 hundred million chronic hepatitis B virus infected people all over the world die of terminal liver diseases caused by HBV, including liver failure, liver cirrhosis and hepatocellular carcinoma ^[1-3] . About 30% of patients with liver cirrhosis are caused by HBV, and about 40% of patients with liver cell carcinoma (HCC) are caused by HBV ^[4] . Hepatitis b virus infection remains a significant public health problem worldwide. However, there is still no effective strategy for treating chronic hepatitis B, and the existing HBV treatment modalities mainly include antiviral drugs (nucleoside/nucleotide analogs) and interferon, which is one of the main treatments for chronic hepatitis BAlthough they have certain therapeutic effects, they usually cannot induce effective immune response, and thus cannot completely eliminate HBV infection; moreover, the side effects caused by long-term administration are large, and the antiviral drugs can also generate drug resistance. Chronic HBV infection is one of main diseases threatening human health, the search for effective immunotherapy strategies for chronic hepatitis B is urgent, and the development of therapeutic vaccines for chronic hepatitis B has very important social and economic significance.

Seasonal influenza can cause 1-4 million people to develop serious illness and 20-50 million people to die each year ^[5] . The vaccine route is the best way to prevent and control influenza, and vaccines can reduce the incidence of disease and can reduce the severity of infection, particularly in young children and the elderly, such at risk groups with influenza complications. Even though the currently approved influenza vaccines are able to produce good protective effects in healthy young adults, there are still some problems to be solved. For example, some vaccines rely on chick embryos for their production, such as inactivated influenza vaccines and attenuated influenza vaccines, which have the disadvantage that if the circulating strain is of avian origin, the disease circulation leads to an increased demand for vaccine and chick embryos, which may lead to problems in the supply of chick embryos ^[6] . Another disadvantage is that the production of these vaccines requires a significant amount of time. The elderly are more likely to develop severe syndromes with influenza virus, and standard vaccines are generally less effective for the elderly, who have a gradually weakened immune system with age ^[7] . In view of the problems encountered by the current influenza vaccines, aiming at influenza virus epidemics, the current influenza vaccine which has stronger immunogenicity, does not depend on chick embryos and can be quickly produced is urgently needed.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pathogen causing a 2019 coronavirus disease (COVID-19) pandemic, and clinical symptoms caused by SARS-CoV-2 mainly include asymptomatic infection, mild influenza-like symptoms, pneumonia and severe acute respiratory distress syndrome, and in severe cases, death of infected patients is caused. Now for the new modelNo specific medicine exists in coronavirus, and the vaccine is a basic strategy for controlling and stopping new coronary pandemic ^[9] . In addition, the emergence of new coronavirus mutants poses new challenges to existing vaccine candidates and to the control of epidemics ^[10] Thus, a vaccine that is potent and can also act on new coronavirus mutants is urgently needed under current epidemic conditions.

When the antigen is linked to the immunoglobulin Fc region, the half-life of the antigen is significantly extended, and the immunoglobulin Fc region can bind to the Fc receptor on the surface of the antigen-presenting cell to facilitate antigen processing and presentation by the antigen-presenting cell ^[11-13] . Type I interferons have a number of biological activities as an antiviral cytokine, one of which involves stimulation of immune cells ^[14] . IFN alpha can powerfully induce the differentiation and activation of human DC cells ^[15] . After acting on immature DCs, type I interferon can promote the expression of MHC molecules and costimulatory molecules on the cell surface of DCs, such as: MHC class I, CD80 and CD86, thereby enhancing the ability of DCs to activate T cells ^[16-18] . Type I interferons have been reported to promote antigen presentation by DCs following vaccinia virus and lymphocytic chorioencephalitis virus (LCMV) infection ^[19-21] . In addition, after acting on DCs, the type I interferon can promote the DCs to migrate to lymph nodes by up-regulating the expression of chemokine receptors, thereby promoting the activation of T cells ^[22，23] . More and more recent studies have shown that type I interferons can be used as immunological adjuvants. Le Bon et al showed that when mice were immunized with weak immunogens, type I interferons exhibited strong immunoadjuvant effects in mice that induced the production of long-lasting antibodies and immunological memory ^[24] The authors also found that the main cell group in which type I interferons act was DC cells. Meanwhile, the antibody is utilized to deliver the vaccine to the DC in a targeted mode, the DC activation and cross presentation functions are stimulated, and the activity and the titer of the vaccine are further enhanced.

The present invention exists a need to provide a vaccine platform that enhances the body's response to viral, bacterial or tumor antigens.

Disclosure of Invention

Vaccines are effective means of preventing and controlling major outbreaks, and there are various types of vaccines, an important one of which is protein subunit vaccines. Generally, simple protein subunit vaccines are generally poor in immunogenicity, which often limits the use of protein subunit vaccines. Therefore, a general protein subunit vaccine platform is highly urgent. Based on the effect of immunoglobulin Fc region and type I interferon on the immune system, the inventors specifically propose interferon alpha-viral antigen, bacterial or tumor-immunoglobulin Fc region fusion protein vaccine platforms to enhance the body's response to viral, bacterial or tumor antigens. The invention provides an I-type interferon-protein antigen-immunoglobulin Fc vaccine platform, wherein the I-type interferon can act on antigen presenting cells to ensure that the antigen presenting cells mature and migrate so as to better play the roles of antigen presenting and activating T cells, and on the other hand, the Fc part of the vaccine platform can be combined with an Fc receptor on the surface of the antigen presenting cells so as to not enhance the antigen uptake of the antigen presenting cells and further help the antigen presenting cells to play roles. The inventor proposes that the fusion of Th cell helper epitopes can further improve the immune response effect of the I-type interferon-protein antigen-immunoglobulin Fc vaccine, and the fusion is an important component of the vaccine. The inventor proposes that the Fc can be replaced by anti-PD-L1 and other antibodies, the vaccine can be delivered to the DC in a targeted manner, the activation and cross presentation functions of the DC are stimulated, and the activity and the potency of the vaccine can be further enhanced. The invention can be used as a novel vaccine platform to be used as a preventive and therapeutic vaccine for diseases such as virus infection, bacterial infection or tumor.

In some embodiments, the present invention provides a vaccine comprising a fusion protein (Th-epitope-attached) of interferon-target antigen-immunoglobulin Fc region (or antibody). In some embodiments, the invention also provides the use of a fusion protein (Th-epitope tag) comprising an interferon-target antigen-immunoglobulin Fc region (or antibody) for the preparation of a prophylactic or therapeutic composition or kit (e.g., a pharmaceutical or vaccine composition or kit). The vaccine of the present invention can be produced by a eukaryotic cell expression system and can be inoculated by an immunization route such as subcutaneous/intramuscular or nasal cavity. The fusion polypeptide of the present invention, wherein the antibody (Ab) is not particularly limited, can include, for example, a whole antibody or a fragment of an antibody, such as heavy and light chains of an antibody, or a single-chain antibody, and can be a DC-targeted activated antibody, including antibodies such as anti-PD-L1, anti-DEC205, anti-CD80/86, etc.

In some embodiments, the target antigen described herein is not particularly limited and can be any suitable antigen. In some embodiments, the target antigen described herein can be, for example, a tumor antigen and/or a pathogen antigen (e.g., a viral or bacterial antigen). In some embodiments, the target antigen described herein can be, for example, a tumor antigen, such as a tumor cell high expression protein molecule, e.g., human Epidermal growth factor receptor 2 (herr 2/neu), epidermal Growth Factor (EGFR).

In some embodiments, the target antigen used in the vaccines provided herein can be, for example, a mutated target antigen that is different from the wild-type. In some embodiments, the target antigen described herein can be, for example, a mutant of a tumor antigen and/or a pathogen antigen (e.g., a viral or bacterial antigen). In some embodiments, the target antigen can be, for example, the full length or S1 region of the S protein of SARS-COV-2 virus, e.g., the target antigen can be the antigen set forth in SEQ ID No.76 or SEQ ID No. 77. In this context, a wild-type target antigen refers to a protein expressed by a virus or other infectious agent or tumor encoded by a wild-type gene, which is the predominant allele in nature and is often used as a standard control gene in biological experiments, and has immunogenicity, such as the Spike protein (S protein) derived from the original wild-type strain of SARS-CoV-2. In this context, mutated target antigens (mutants) refer to mutant viral proteins expressed by mutant viral strains encoded by mutant genes mutated from the wild-type gene, e.g. point mutations found in the S protein of different mutant neocoronaviruses comprise: NTD 69-70 deletion, Y144 deletion, 242-244 deletion, L18F, D80A, D215, R246I mutation, RBD region K417 mutation, E484 mutation, N501Y mutation, L452R mutation, T478K mutation, D614G mutation, H655Y mutation. For example, these point mutations are present in different combinations in mutant neocoronavirus derived from British B.1.1.7 (Alpha) mutants, south African B.1.351 (Beta) mutants, brazil P1 (Gamma) mutants, india B.1.617, B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.617.3 mutants, calif. B.1.429 mutants, and the like. In some embodiments, the mutated target antigen may include, for example, a natural point mutation/deletion mutation/gain mutation/truncation, an artificial point mutation/deletion mutation/gain mutation/truncation, any combination of natural or artificial mutations, a subtype generated after mutation, wherein the target antigen may be a tumor antigen, a pathogen antigen such as a viral (e.g., SARS-COV-2) or bacterial antigen. In some embodiments, the target antigen used in the vaccines provided herein is a mutated viral antigen, e.g., the mutated viral antigen can be a mutant of SARS-COV-2, including, e.g., a natural point mutation/deletion mutation/gain mutation/truncation of the SARS-COV-2 protein (e.g., one or more of the S protein, N protein, M protein, E protein), an artificial point mutation/deletion mutation/gain mutation/truncation, any combination of natural or artificial mutations, a subtype produced after mutation, e.g., the mutated viral antigen can be a mutant of the full length S protein, S1 region, RBD region, e.g., the mutated viral antigen can comprise one or more of the following mutations of the S protein of SARS-COV-2: NTD regions 69-70 deletion, Y144 deletion, 242-244 deletion, L18F, D80A, D215, R246I mutation, RBD regions K417, E484, N501Y mutation, L452R mutation, T478K mutation, D614G, H655Y mutation, for example the viral antigen of the mutation may comprise a mutation present in a mutant derived from b.1.1.7 (Alpha) mutant, south african b.1.351 (Beta) mutant and brazil P1 (Gamma) mutant, indian b.617.617, b.1.1.1 (Kappa), b.1.617.2 (Delta), b.1.617.3 mutant, ca b.1.429 mutant, for example the mutated viral antigen may comprise a mutant of any one of SEQ ID No.79, SEQ ID No.80, SEQ ID No.81, for example the mutated viral antigen may be any one of SEQ ID No.79, SEQ ID No.80, SEQ ID No.81, SEQ ID No.84. Herein, reference to a target antigen herein generally includes both wild-type target antigens and mutant target antigens, unless otherwise specifically indicated or the context clearly limits.

The object of the present invention is to provide a vaccine platform consisting of Interferon (IFN) and tumor, bacterial or viral antigens (hepatitis b virus Pres1 antigen, SARS-COV 2RBD antigen, influenza HA antigen, human papilloma virus HPV E7 antigen, hepatitis b virus surface antigen (HBsAg) or peptide fragments, herpes zoster virus (VZV) gE antigen, ebostein-Barr virus (EBV) EBNA1/LMP2/gp350, herpes simplex virus 2 (HSV-2) gD antigen, HIV gp120 antigen-immunoglobulin Fc region (or antibody) (additional Th epitope) said fusion protein may be a homologous or heterologous dimeric protein when said fusion protein is a dimer, the interferon, target antigen, immunoglobulin Fc region (or antibody) as a structural unit may be present in the first polypeptide chain and/or the second polypeptide chain, the presence of each structural unit is not particularly limited, e.g. may be present in one chain at the same time, and one or more structural units may be present in any one chain, and one or more structural units may be present in another chain.

The interferons of the invention may be selected from the group consisting of type I interferons, type II interferons and type III interferons, such as IFN- α, IFN- β, IFN- γ, IFN- λ 1 (IL-29), IFN- λ 2 (IL-28 a), IFN- λ (IL-28 b), and IFN- ω; the IFN may be from human or murine origin; type I interferon IFN-. Alpha.s (SEQ ID NO.1, SEQ ID NO.21, SEQ ID NO. 22) are preferred.

The immunoglobulin Fc region according to the invention can be selected from the constant region amino acid sequences of IgG1, igG2, igG3 and IgG4 and/or IgM, preferably IgG1 (SEQ ID NO.2, SEQ ID NO.23, SEQ ID NO. 24).

The fusion polypeptide of the invention may also optionally comprise one or more Th cell helper epitopes and/or linker fragments (linkers). For example, when the fusion protein is a dimer, optionally the fusion protein may also comprise one or more Th cell helper epitopes and/or connecting fragments in either or both chains (i.e., the first polypeptide chain and/or the second polypeptide chain) of the homodimer or heterodimer. As known to the person skilled in the art, the individual building blocks of the fusion protein can be connected by means of suitable connecting segments (linkers). The linking fragment that can be used in the vaccine of the present invention is not particularly limited and may be any suitable peptide fragment known in the art. The connecting segment of each structural unit can be a flexible polypeptide sequence, and can be connecting

segments

1 and 2, such as shown in SEQ ID NO.4 and SEQ ID NO.25 amino acid sequences.

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

Preferred antigens described herein include hepatitis B Pres1 antigen, including the ad subtype (SEQ ID NO. 6) ay subtype (SEQ ID NO. 26), HBV HBsAg antigen (various subtypes and peptides), including the adr subtype (SEQ ID NO. 7), adw subtype (SEQ ID NO. 27), ayw subtype (SEQ ID NO. 28), SARA-COV 2RBD antigen (SEQ ID NO. 8), influenza HA antigen (SEQ ID NO. 9), HPV E7 antigen (SEQ ID NO. 10). Herpes virus VZV-gE antigen (SEQ ID NO. 91), EBV-gp350 antigen (SEQ ID NO. 92), HSV-2-gD antigen (SEQ ID NO. 93).

The homodimer protein described in the invention comprises a first polypeptide and a second polypeptide, and the first polypeptide and the second polypeptide are completely identical. The first polypeptide and the second polypeptide are IFN-tumor or virus antigen (hepatitis B Pres1 antigen, SARS-COV 2RBD 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 in sequence from the N section to the C section; or a polypeptide comprising a Pan epitope. 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 and 68.

The heterodimer comprises a first polypeptide and a second pair of peptides, wherein the first polypeptide and the second polypeptide are not the same polypeptide, the first polypeptide is respectively an IFN-immunoglobulin Fc region from a C section to an N end and comprises amino acid sequences shown in SEQ ID NO.15, 33, 42, 51, 60 and 69; the second polypeptide is tumor or virus antigen (hepatitis B Pres1 antigen, SARS-COV 2RBD 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 section C to section N; comprises the amino acid sequence shown in SEQ ID NO.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.

The invention also provides an amino acid sequence of a Pres1 antigen, an HBsAg antigen or a peptide segment, a SARS-COV 2RBD antigen, an influenza HA antigen, an HPV E7 antigen, a VZV-gE antigen, an EBV EBNA1/LMP2/gp350, an HSV-2-gD antigen and an HIV gp120 antigen-immunoglobulin Fc vaccine platform for coding the IFN-tumor or virus antigen.

The invention also relates to nucleotide fragments encoding the vaccine platforms, fusion proteins.

The invention also relates to a preparation method of the fusion protein or vaccine platform, for example, the preparation method comprises the following steps:

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

(2) Constructing a host cell comprising said expression vector by transient transfection of a host cell, preferably said host cell is a 293F cell;

(3) Culturing the host cell and collecting the cell supernatant;

(4) The fusion protein or vaccine platform was purified by protein purification on an affinity column of ProteinA/G.

The invention also comprises the application of the vaccine platform, the vaccine platform can be used as a hepatitis B preventive vaccine, the vaccine platform is used as a hepatitis B therapeutic vaccine, the vaccine platform is used as an influenza preventive vaccine, the vaccine platform is used as a SARA-COV2, influenza, HPV, VZV, EBV, HSV-2 and HIV preventive vaccine, and the vaccine platform is used as an HPV and EBV related tumor preventive vaccine.

The invention comprises an 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.

The vaccine platform is used as an HBV therapeutic vaccine combined with a hepatitis B virus envelope protein HBsAg vaccine for clinical use in the chronic hepatitis B virus infection treatment process.

The invention comprises the vaccine platform as an HBV therapeutic vaccine combined with nucleoside or nucleotide the analogue is clinically used in the treatment process of chronic hepatitis B virus infection.

The invention comprises the combined application of the vaccine platform as HBV, influenza, SARA-COV2, HPV, VZV, EBV, HSV-2, HIV preventive or therapeutic vaccine and the like, and antiviral drugs and other treatment methods; the combined application of HBV, HPV and EBV related tumor preventive or therapeutic vaccine and antiviral and antitumor drug and therapy.

The invention comprises the vaccine platform as a component of a multivalent combined vaccine which is combined with other virus or pathogen or tumor vaccines.

The vaccine comprises any one fusion protein vaccine of the vaccine platform and adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of the same virus, pathogen or tumor, which are immunized according to a sequential or simultaneous immunization program.

The invention encompasses the full-length sequence and any truncation sequence of the vaccine platform antigen, such as SEQ ID NO.76, SEQ ID NO.77, SEQ ID NO.78.

Any mutant which may appear in the vaccine antigen containing the fusion protein comprises natural point mutation/deletion mutation/truncation body, any combination of natural point mutation, subtype generated after mutation, artificial point mutation/deletion mutation/truncation body and other mutant sequences which are constructed by the invention for enhancing 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.

Any vaccine of the present invention as a component of a vaccine, such as a multivalent combined vaccine consisting of another vaccine of the present invention or another vaccine different from the vaccine of the present invention, such as other virus or pathogen or tumor vaccine, for example, a multivalent vaccine combining the SARS-COV-2 fusion protein vaccine of the present invention with influenza vaccine or other vaccine, for example, any vaccine of the present invention is immunized with the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of the same virus, pathogen, tumor according to a sequential or simultaneous immunization program, for example, the SARS-COV-2 fusion protein vaccine is immunized with the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of SARS-COV-2 according to a sequential or simultaneous immunization program, for example, the sequential immunization sequence may be: 1) Firstly, the SARS-COV-2 fusion protein vaccine is immunized, and then the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of SARS-COV-2 is immunized; 2) Firstly, immunizing adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of SARS-COV-2, and then immunizing ARS-COV-2 fusion protein vaccine; 3) The SARS-COV-2 fusion protein vaccine and SARS-COV-2 adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine are simultaneously immunized. When used in combination, the combination vaccine can be prepared as a convenient kit, as is known in the art.

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

1. the antigen of the IFN-tumor or virus antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the invention can be changed by various components, and can be a tumor-associated antigen or a virus-specific antigen, so that the use flexibility of the vaccine platform is enhanced, and the use range of the vaccine platform is also enhanced.

2. The IFN-tumor or virus antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the invention has the advantages that Interferon (IFN) can enhance migration and maturation of antigen presenting cells, so that co-stimulatory molecules expressed by the antigen presenting cells are increased, and antigen presentation to T cells is facilitated, meanwhile, an Fc region (or antibody) in the vaccine platform enhances the molecular weight of the antigen so as to increase the half-life of the antigen, and on the other hand, the Fc region (or antibody) can be combined with an Fc receptor on the surface of the antigen presenting cells so as to promote processing and presentation of the antigen by the antigen presenting cells, and further facilitate generation of immune response.

3. The IFN-tumor or virus antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the invention is expressed by a eukaryotic HEK293 cell expression system, and the protein expressed by the HEK293 cell is more similar to a natural protein molecule in molecular structure, physicochemical characteristics, protein modification and biological functions of the protein.

4. The IFN-tumor or virus antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the invention has two structures of homologous or heterologous dimers, and is more excellent to select different antigens.

5. The IFN-tumor or virus antigen-immunoglobulin Fc vaccine platform provided by the invention can activate DC to enhance DC cross presentation and generate strong B cell and T cell immune response by fusing Th cell auxiliary epitopes, such as Pan epitopes, utilizing DC targeting antibodies such as anti-PD-L1 and the like and adding various adjuvants for stimulating immune response.

6. The IFN-tumor or virus antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the invention has a wide application range, and can be used as a preventive vaccine and a therapeutic vaccine.

7. The IFN-tumor or virus antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the invention can be used alone, and can also be used as a therapeutic vaccine in combination with the existing HBsAg commercial vaccine and nucleoside/nucleotide analogues.

8. The vaccine platform provided by the invention can be used as a component of a vaccine to form a multivalent combined vaccine with other viruses or pathogens or tumor vaccines.

9. Any one of the fusion protein vaccines in the vaccine platform provided by the invention can be used for immunizing with adenovirus vaccines or mRNA vaccines or inactivated vaccines or DNA vaccines of the same virus, pathogen or tumor according to a sequential or simultaneous immunization program.

10. The full-length sequence and any truncation sequence of the vaccine platform antigen provided by the invention.

11. Any mutant which may appear in the vaccine platform antigen provided by the invention comprises a natural point mutation/deletion mutation/augmentation mutation/truncation body, any combination of natural point mutations, subtypes generated after mutation, and artificial point mutation/deletion mutation/augmentation mutation/truncation body and other mutation sequences which are constructed by the invention for enhancing the vaccine effect.

Sequence information referred to herein:

1. sequence of unit building blocks:

SEQ ID NO.1: mouse mIFN alpha 4 amino acid sequence (mIFN alpha)

SEQ ID No.21: human IFN alpha 2 amino acid sequence (hIFN alpha)

SEQ id No.22: human mutant IFN alpha 2 (Q124R) amino acid sequence (hmIFN alpha)

SEQ ID NO.2: human IgG1-Fc amino acid sequence

SEQ ID No.23: heterodimeric Fc-hole

SEQ ID No.24: heterodimeric Fc-knob

SEQ ID NO.3: amino acid sequence of Th helper epitope Pan HLA-binding epitope (PADER)

SEQ ID No.4: linker 1 amino acid sequence:

SEQ ID No.25: linker 2 amino acid sequence:

SEQ ID No.5: signal peptide amino acid sequence:

SEQ ID No.6: amino acid sequence of HBV Pres1 (ad subtype)

SEQ ID NO.26: amino acid sequence of HBV Pres1 (ay subtype)

SEQ ID NO.7: amino acid sequence of HBV HBsAg (adr subtype)

SEQ ID NO.27: amino acid sequence of HBV HBsAg (adw subtype)

SEQ ID No.28: amino acid sequence of HBV HBsAg (ayw subtype)

SEQ ID No.8: amino acid sequence of SARS-CoV-2RBD

SEQ ID NO.9: HA amino acid sequence of influenza virus

SEQ ID No.10: amino acid sequence of HPV-E7 antigen

2. Murine IFN vaccine mfna α -antigen-Fc sequence:

SEQ ID NO.11: mIFN alpha-Pres 1-Fc amino acid sequence in homodimer

SEQ ID No.12: mIFN alpha-RBD (SARS-CoV-2) -Fc amino acid sequence in homodimer

SEQ ID No.13: mIFN alpha-HA-Fc amino acid sequence in homodimer

SEQ ID No.14: mIFN alpha-E in homodimers 7 (HPV) -Fc amino acid sequence

SEQ ID NO.15: first chain mIFN alpha-Fc-hole amino acid sequence in heterodimer

SEQ ID No.16: amino acid sequence of the second chain Pres 1-Fc-kb of heterodimer mIFN alpha-Pres 1-Fc

SEQ ID NO.17: amino acid sequence of second chain RBD (SARS-CoV-2) -Fc-knob in heterodimer mIFN alpha-RBD (SARA-CoV-2) -Fc

SEQ ID NO.18: amino acid sequence of second chain HA-Fc-knob in heterodimer mIFN alpha-HA-Fc

SEQ ID NO.19: amino acid sequence of the second chain E7-Fc-knob in the heterodimer mIFN alpha-E7 (HPV) -Fc

3. Murine IFN vaccine IFN α -Pan-antigen-Fc sequence containing a Pan epitope:

SEQ ID NO.29: mIFN alpha-Pan-Pres 1-Fc amino acid sequence in homodimer

SEQ ID No.30: mIFN alpha-Pan-RBD (SARS-CoV-2) -Fc amino acid sequence in homodimer

SEQ ID NO.31: mIFN alpha-Pan-HA-Fc amino acid sequence in homodimer

SEQ ID NO.32: mIFN alpha-Pan-E7 (HPV) -Fc amino acid sequence in homodimer

SEQ ID NO.33: first chain mIFN alpha-Fc-hole amino acid sequence in heterodimer

SEQ ID No.34: amino acid sequence of second chain Pan-Pres1-Fc-knob in heterodimer mIFN-Pan-Pres1-Fc

SEQ ID NO.35: amino acid sequence of second chain Pan-RBD (SARS-CoV-2) -Fc-knob in heterodimer mIFN alpha-Pan-RBD (SARA-CoV-2) -Fc

SEQ ID NO.36: second chain Pan-HA-Fc-knob amino acid sequence in heterodimer mIFN alpha-Pan-HA-Fc

SEQ ID No.37: second chain Pan-E7-Fc-knob amino acid sequence in heterodimer mIFN alpha-Pan-E7 (HPV) -Fc

4. Human IFN vaccine hIFN alpha-antigen-Fc sequence:

SEQ ID NO.38: hIFN alpha-Pres 1-Fc amino acid sequence in homodimer

SEQ ID NO.39: hIFN alpha-RBD (SARS-CoV-2) -Fc amino acid sequence in homodimer

SEQ ID No.40: hIFN alpha-HA-Fc amino acid sequence in homodimer

SEQ ID NO.41: hIFN alpha-E7 (HPV) -Fc amino acid sequence in homodimer,

SEQ ID No.42: first chain hIFN-Fc-hole amino acid sequence in heterodimer

SEQ ID No.43: amino acid sequence of second chain Pres1-Fc-knob in heterodimer hIFN alpha-Pres 1-Fc

SEQ ID NO.44: amino acid sequence of second chain RBD (SARS-CoV-2) -Fc-knob in heterodimer hIFN alpha-RBD (SARA-CoV-2) -Fc

SEQ ID No.45: amino acid sequence of second chain HA-Fc-knob in heterodimer hIFN alpha-HA-Fc

SEQ ID No.46: amino acid sequence of the second chain E7 (HPV) -Fc-knob in heterodimer hIFN alpha-E7 (HPV) -Fc

5. Human IFN vaccine IFN alpha-Pan-antigen-Fc sequence containing Pan epitope:

SEQ ID No.47: hIFN alpha-Pan-Pres 1-Fc amino acid sequence in homodimer

SEQ ID No.48: hIFN alpha-Pan-RBD (SARS-CoV-2) -Fc amino acid sequence in homodimer

SEQ ID NO.49: hIFN alpha-Pan-HA-Fc amino acid sequence in homodimer

SEQ ID No.50: hIFN alpha-Pan-E7 (HPV) -Fc amino acid in homodimer

SEQ ID NO.51: first chain hIFN alpha-Fc-hole amino acid sequence in heterodimer

SEQ ID No.52: heterodimer hIFN alpha-Pan-Pres 1-Fc second two-chain Pan-Pres1-Fc-knob amino acid sequence

SEQ ID No.53: amino acid sequence of second chain Pan-RBD (SARS-CoV-2) -Fc-knob in heterodimer hIFN alpha-Pan-RBD (SARA-CoV-2) -Fc

SEQ ID No.54: second chain Pan-HA-Fc-knob amino acid sequence in heterodimer hIFN alpha-Pan-HA-Fc

SEQ ID No.55: amino acid sequence of second chain Pan-HA-Fc-knob in heterodimer hIFN alpha-Pan-E7 (HPV) -Fc

6. Human mutant IFN vaccine hmIFN α -Pan-antigen-Fc sequence:

SEQ ID NO.56: hmIFN alpha-Pres 1-Fc amino acid sequence in homodimer

SEQ ID No.57: hmIFN alpha-RBD (SARS-CoV-2) -Fc amino acid sequence in homodimer

SEQ ID No.58: hmIFN alpha-HA-Fc amino acid sequence in homodimer

SEQ ID No.59: hmIFN alpha-E7 (HPV) -Fc amino acid sequence in homodimer

SEQ ID No.60: first chain hmIFN-Fc-hole amino acid sequence in heterodimer

SEQ ID No.61: second chain Pres 1-Fc-kb amino acid sequence in heterodimer hmIFN alpha-Pres 1-Fc

SEQ ID No.62: amino acid sequence of second chain RBD (SARS-CoV-2) -Fc-knob in heterodimer hmIFN alpha-RBD (SARA-CoV-2) -Fc

SEQ ID No.63: second chain HA-Fc-knob amino acid sequence in heterodimer hmIFN alpha-HA-Fc

SEQ ID NO.64: second chain HA-Fc-knob amino acid sequence in heterodimer hmIFN alpha-E7 (HPV) -Fc

7. Humanized mutant IFN (human interferon) Pan epitope-containing vaccine hmIFN alpha-Pan epitope-antigen-Fc sequence

SEQ ID No.65: hmIFN alpha-Pan-Pres 1-Fc amino acid sequence in homodimer

SEQ ID NO.66: hmIFN alpha-Pan-RBD (SARS-CoV-2) -Fc amino acid sequence in homodimer

SEQ ID NO.67: hmIFN alpha-Pan-HA-Fc amino acid sequence in homodimer

SEQ ID No.68: hmIFN alpha-Pan-E7 (HPV) -Fc amino acid sequence in homodimer

SEQ ID NO.69: first chain hmIFN alpha 4-Fc-hole amino acid sequence in heterodimer

SEQ ID No.70: amino acid sequence of second chain Pan-Pres1-Fc-knob in heterodimer hmIFN alpha-Pan-Pres 1-Fc

SEQ ID NO.71: amino acid sequence of second chain Pan-RBD (SARS-CoV-2) -Fc-knob in heterodimer hmIFN alpha-Pan-RBD (SARA-CoV-2) -Fc

SEQ ID No.72: second chain Pan-HA-Fc-knob amino acid sequence in heterodimer hmIFN alpha-Pan-HA-Fc

SEQ ID NO.73: amino acid sequence of second chain Pan-HA-Fc-knob in heterodimer hmIFN alpha-Pan-E7 (HPV) -Fc

8. Fc-substituting antibody sequences

SEQ ID No.20: scFv (PD-L1) amino acid sequence

SEQ ID No.74: anti-PD-L1 VH amino acid sequence

SEQ ID No.75: anti-PD-L1 VL amino acid sequence

9. Other viral antigen sequences

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

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

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

Amino acid sequence of SARS-CoV-2 British mutant (B.1.1.7, alpha) RBD protein of SEQ ID NO.79

Amino acid sequence of SEQ ID NO.80 SARS-CoV-2 south African mutant (B.1.351, beta) RBD protein

Amino acid sequence of SEQ ID NO.81 SARS-CoV-2 Brazilian mutant strain (P1) RBD protein

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

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

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

10. Other tumor antigen sequences:

the amino acid sequences of murine Her2 extracellular domain II, III and IV referred to in the examples:

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

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

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

amino acid sequences of extracellular domains II, III and IV of human Her2 referred to in the examples:

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

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

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

11. herpesvirus antigen sequences referred to in the examples:

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

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

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

drawings

FIG. 1 is a schematic diagram of the vaccine platform in the form of homodimers, arranged and combined in the interferon-linker 1-target antigen-immunoglobulin Fc (or antibody) order;

FIG. 2 schematic representation of the vaccine platform in the form of heterodimers, according to the interferon-linked fragment 1-IgG1-hole, target antigen-IgG 1-knob (or antibody) combination;

FIG. 3 schematic representation of the vaccine platform in the form of heterodimers, according to interferon-linked fragment 1-IgG1-knob combination and target protein IgG1-hole (or antibody), respectively;

FIG. 4 is a schematic diagram of the vaccine platform in the form of homodimers, sequentially arranged and combined according to the interferon-linker 1-Th cell helper epitope-linker 2-target antigen-immunoglobulin Fc (or antibody);

FIG. 5 schematic representation of the vaccine platform in the form of heterodimers, according to interferon-linker 1-IgG1-hole and Th cell helper epitope-linker 2-target antigen-IgG 1-knob (or antibody) combinations, respectively;

FIG. 6 schematic representation of the vaccine platform in the form of heterodimers, according to interferon-linker 1-IgG1-knob and Th cell helper epitope-linker 2-target antigen-IgG 1-hole (or antibody) combinations, respectively.

FIG. 7 SDS-PAGE identification of Pres1-Fc and IFN-Pres1-Fc non-denatured proteins

FIG. 8 fusion proteins preS1-Fc and IFN-preS1-Fc significantly enhance the immunogenicity of antigenic molecules compared to free preS1 and lead to the production of a broad spectrum of neutralizing antibodies. (a) C57/BL6 (n = 8/group) mice were immunized subcutaneously with free hepatitis B Pres1, pres1-Fc, IFN α -Pres1-Fc protein and the level of Pres 1-specific antibodies in serum was measured at the indicated times by the method of Elisa. (b) Three HBV genotypes stably carrying mice (n = 4) serum from IFN α -Pres1-Fc protein immunized mice was injected by intravenous injection, and changes in Pres1 antigen in the serum were detected after 12 hours.

FIG. 9 IFN alpha Pres1-Fc can be applied as a preventive vaccine against hepatitis B. C57/BL6 mice were immunized subcutaneously with free hepatitis B Pres1, pres1-Fc, IFN α -Pres1-Fc protein, and were infected with 1x10 by tail vein injection 28 days after inoculation ¹¹ vg of AAV-HBV1.3 virus. (a) Anti-Pres1 levels in serum before and at

weeks

1, 2, 3, 4 after virus inoculation. (b) detecting the level of Pres1 in serum at the indicated time points. (c) Serum HBsAg levels were measured by Elisa at

weeks

1, 2, 3, and 4. (d) Proportion of HBsAg positive mice after AAV-HBV1.3 virus inoculation.

Figure 10 IFN alpha Pres1-Fc as chronic infection of B therapeutic vaccine. C57/BL6 mice were infected with 1X10 by tail vein injection ¹¹ vg AAV-HBV1.3 virus, after 6 weeks of infection, stably infected mice (n = 8/group) are selected, and are inoculated with recombinant Pres1 and IFN alpha-Pres 1-Fc protein in a subcutaneous immunization mode, and are immunized once every 2 weeks of isolation for three times. (a) detection of Anti-Pres1 antigen in serum; (b) detection of Pres1 antigen in serum; (c) Detection of level of HBV-associated antigen HBsAg in mouse serum

FIG. 11 Th cell helper epitopes enhance the antibody response of IFN α -Pres1-Fc vaccine

Compared with IFN-preS1-Fc, IFN-Pan-preS1-Fc can obviously enhance the immunogenicity of the antigen molecule. C57/BL6 (n = 8/group) mice were inoculated with hepatitis b Pres1, pres1-Fc, IFN α -Pres1-Fc proteins without aluminum adjuvant by subcutaneous immunization, and the level of Pres 1-specific antibodies in serum was measured at the indicated time using the method of Elisa.

FIG. 12 IFN alpha Pan Pres1-Fc as chronic infection of B type therapeutic vaccine. C57/BL6 mice were infected with 1x10 by tail vein injection ¹¹ vg AAV-HBV1.3 virus, after 6 weeks of infection, stably infected mice (n = 8/group) are selected, and are inoculated with recombinant Pres1 and IFN alpha-Pres 1-Fc protein in a subcutaneous immunization mode, and are immunized once every 2 weeks of isolation for three times. (a) detection of Anti-Pres1 antigen in serum; (b) detection of Pres1 antigen in serum; (c) detecting the level of HBV-associated antigen HBsAg in mouse serum; (d) By QPCRThe level of HBV-DNA in the serum of mice was measured.

FIG. 13 IFN α -Pres1-Fc in combination with commercial HBsAg vaccine, breaks immune tolerance against HBsAg and induces HBsAg-HBsAb serological switch. HBV Carrier mice were immunized subcutaneously with IFN α -Pres1-Fc and HBsAg commercial vaccines three times in a total of once every two weeks. (a) the level of Pres1 in serum in HBV Carrier mice, (b) the level of HBsAg, (c) the level of Anti-Pres1 in serum (d), the level of Anti-HBsAg in serum (e) the level of HBV-DNA in serum. * P < 0.001

FIG. 14 IFN α -RBD (SARS-CoV 2) -Fc elicits a stronger antibody response than free SARS-Cov 2RBD protein. Balb/c (n = 8/group) mice were immunized subcutaneously with free SARS-Cov-2RBD, RBD-Fc, IFN α -RBD-Fc protein, and the level of SARS-Cov 2S protein-specific antibodies in serum was measured by Elisa at the indicated time. * P < 0.0001.

FIG. 15 IFN alpha RBD (SARS-CoV 2) -Fc immune mice after the generation of high titer antiviral serum, in vitro cell experiments, can completely block SARS-CoV2 pseudovirus infection.

FIG. 16 detection of antiserum RBD-specific antibodies generated by IFN α -Pan-RBD (original strain) -Fc and IFN α -Pan-RBD (SARS-CoV-2 south Africa mutant) -Fc immunization. (a) IFN alpha-Pan-RBD (SARS-CoV-2 original strain) -Fc SDS-PAGE electrophoresis identification picture. (b) IFN alpha-Pan-RBD (SARS-CoV-2 south Africa mutant strain) -Fc SDS-PAGE identification picture. (c) IFN alpha-Pan-RBD (original strain) -Fc and IFN alpha-Pan-RBD (SARS-CoV-2 south Africa mutant) -Fc immune after 14 days in serum RBD specific antibody and original strain RBD binding situation. (d) IFN alpha-Pan-RBD (original strain) -Fc and IFN alpha-Pan-RBD (SARS-CoV-2 south African mutant) -Fc immune after 14 days in serum RBD specific antibody and south African mutant RBD binding situation.

FIG. 17 (a) SDS-PAGE electrophoresis identification profiles of Mouse IFN α -RBD-Fc and Mouse IFN α -Pan-RBD-Fc proteins. (b) SDS-PAGE electrophoresis identification maps of Human IFN alpha-RBD-Fc and Human IFN alpha-Pan-RBD-Fc proteins.

FIG. 18 Pan (Pan DR-binding epitope) CD 4T cell helper epitope can further enhance the immunogenicity of IFN α -RBD-Fc. Mice were vaccinated intramuscularly with 10 μ g Mouse IFN α -RBD-Fc, mouse IFN α -Pan-RBD-Fc or 10 μ g Human IFN α -RBD-Fc, human IFN α -Pan-RBD-Fc protein, and a booster immunization was performed 14 days after vaccination. Mouse sera were collected at 7, 14, and 28 days after immunization, respectively, and the level of RBD-specific antibodies in the mouse sera was measured by ELISA. * P is less than 0.05; * P < 0.0001.

FIG. 19 aluminum adjuvant can enhance specific humoral immune response induced by Human IFN alpha-RBD-Fc, human IFN alpha-Pan-RBD-Fc protein. C57BL/6 mice were inoculated with 10. Mu.g of Human IFN α -RBD-Fc or Human IFN α -Pan-RBD-Fc protein on days 0 and 14 with or without the aid of aluminum adjuvant (AL-), and mouse sera were collected on

days

7, 14, and 28 after inoculation, and the levels of SARS-CoV-2 RBD-specific antibodies in the mouse sera were determined by ELISA. * P is less than 0.05; * P < 0.0001.

FIG. 20.IFN-Pan-RBD-Fc nasal drop immunization induced the production of high titer RBD-specific IgG, igA neutralizing antibody antibodies. The C57BL/6 mice at 6-8 weeks are divided into 5 groups, each group comprises 10 mice, 10 mug of IFN alpha-pan-RBD-Fc or the same number of moles of RBD, RBD-Fc and IFN alpha-RBD-Fc protein are respectively immunized by means of nasal drip immunization, and the volume of nasal drip is 10 uL/mouse. Mice were immunized on days 0 and 14 using two immunization programs. Collecting mouse sera at 7, 14, 21, 28, 35 and 42 days after immunization respectively, and detecting the levels of IgG (a) and IgA (b) of the new crown RBD specific antibodies in each group of sera by using an ELISA method; serum was collected for 42 days and subjected to SARS-CoV-2 pseudovirus in vitro neutralization test (c). The statistical method comprises the following steps: one-way ANOVA, p < 0.05 with significant differences, p < 0.01, p < 0.001, p < 0.0001.

FIG. 21. Non-adjuvant novel corona vaccine IFN-Pan-RBD-Fc nasal drop immunization induces high titer RBD specific IgG, igA neutralizing antibodies in the nasopharynx and lung tissues. After 6-8 weeks C57BL/6 mice were immunized for 28 days, they were sacrificed and their nasal mucosa was removed and disrupted by a tissue homogenate disruptor. And (3) centrifuging the homogenized liquid at 13000rpm for 10 minutes at a high speed, and taking the supernatant as nasal mucosa supernatant (NMDS). Detecting the levels of IgG (a) and IgA (b) of the new crown RBD specific antibodies in each group of nasal mucosa supernatant (NMDS) by adopting an ELISA method; serum was collected for 28 days and subjected to SARS-CoV-2 pseudovirus in vitro neutralization test (c). The statistical method comprises the following steps: one-way ANOVA, p < 0.05 with significant differences, p < 0.01, p < 0.001, p < 0.0001.

FIG. 22. Non-adjuvant neocoronavirus IFN-Pan-RBD-Fc intranasal immunization induced high-titer RBD-specific IgG and IgA antibodies in lung tissue. C57BL/6 mice were sacrificed 28 days after immunization (see FIG. 19) at 6-8 weeks. In the lung of the mouse, about 0.8ml of HBSS +100uMEDTA is sucked by a 1ml syringe, the mouse is injected into an air tube cannula, after gentle blowing and sucking are carried out repeatedly for three times, the liquid is sucked out and collected into a centrifugal tube, and the step is repeated for three times, so that about 2ml of lung lavage liquid can be finally obtained. Centrifuging 500g of mouse lung lavage fluid for 5 minutes to obtain a supernatant, namely the mouse lung lavage fluid (BALF), and detecting the content of new crown RBD specific antibodies IgG (a) and IgA (b) in each group of mouse lung lavage fluid (BALF) by adopting an ELISA method. The statistical method comprises the following steps: one-way ANOVA, p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

Figure 23 her2 vaccine protein expression and purification. 293F cells, SDS-PAGE and Coomassie blue staining to determine the size and purity of the proteins. IFN alpha-3-Fc (62.6 kDa); IFN alpha pan-3-Fc (63.9 kDa); IFN alpha-pan-4-Fc (74.9 kDa) and IFN alpha-4-Fc (73.6 kDa).

FIG. 24 analysis of Her2 vaccine IFN alpha 3-Fc and IFN alpha pan 3-Fc anti-tumor activity. Constructing a TUBO breast cancer model mouse, carrying out administration treatment on the tumor size of 50-80mm < 3 >, and carrying out intratumoral injection on the related fusion protein for 3 times per week. IFN alpha-3-Fc was administered at a dose of 10 ug/dose/mouse, other fusion proteins were administered equimolar, cpG as an adjuvant. Tumor size was measured and tumor growth curves were plotted.

FIG. 25 analysis of IFN α and Pan function to enhance Her2 antigen immunogenicity. BALB/C mice (n = 5) at 6-8 weeks were immunized subcutaneously with the mouse Her2 fusion protein vaccine 4-Fc, IFN α -4-Fc and IFN α -pan-4-Fc without adjuvant. The immunization dose was IFN alpha-4-Fc 10 ug/dose/mouse, other fusion proteins equimolar vaccination. Venous blood was collected 14 days and 21 days after immunization and antibody levels of specific IgG in Her2 sera were measured by ELISA.

FIG. 26 is an SDS-PAGE identification map of IFN-HA1-Fc fusion protein.

Figure 27 mice were vaccinated intramuscularly with 10ug IFN-HA1-Fc or the same molar amount of HA1 protein and a booster immunization was performed 14 days after the primary vaccination. Mouse sera were collected at day 28 after immunization and assayed for HA 1-specific antibody levels by ELISA. Mice were infected with 1000PFU of A/PR8 influenza virus at 42 days post immunization by nasal infection, and mice were observed and their weight changes recorded from the third day post viral infection. (a) Mouse serum was collected 28 days after the primary immunization, and the HA 1-specific antibody level in the mouse serum was measured by ELISA. (b) mouse body weight change following viral infection.

FIG. 28 is an SDS-PAGE identification chart of IFNa-Pan-VZV-gE-Fc, IFNa-Pan-EBV-gp350-Fc, IFNa-Pan-HSV-2-gD-Fc proteins.

FIG. 29 IFN α -Pan-EBV gp350-Fc induces a stronger humoral immune response compared to free EBV gp 350. C57BL/6 (n = 5/group) mice were immunized intramuscularly with 10 μ g IFN α -Pan-EBV gp350-Fc or the same molar amount of EBV gp350 protein and boosted at the same dose 14 days after the primary immunization. At 14 days and 28 days after the primary immunization, mouse serum was collected, and the level of EBV gp 350-specific antibodies in the mouse serum was measured by ELISA.

FIG. 30 IFNa-Pan-HSV-2 gD-Fc elicits a stronger humoral immune response than free HSV-2 gD protein. C57BL/6 (n = 5) mice were vaccinated intramuscularly on days 0 and 14, respectively, with 10 μ g IFNa-Pan-HSV-2 gD-Fc or the same molar amount of HSV-2 gD protein, and mouse sera were collected on days 14 and 28 after the initial vaccination and tested for HSV-2 gD specific antibody levels in the sera by ELISA.

FIG. 31 IFNa-Pan-VZV gE-Fc elicits a stronger humoral immune response and induces a Th1/Th2 balanced T cell immune response than free VZV-2 gE protein.

C57BL/6 (n = 6/group) mice were vaccinated intramuscularly with 10 μ g IFN α -Pan-VZV gE-Fc or the same molar amount of VZV E protein and boosted at the same dose 14 days after the primary immunization. At 14 days and 28 days after the primary immunization, mouse sera were collected and the EBV gp 350-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 mouse sera 28 days after primary immunization.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail below with reference to embodiments and the accompanying drawings. The described embodiments are merely illustrative of the present invention and are not intended to limit the scope of the invention, which is intended to be exemplary of only a portion of the invention rather than the full range of embodiments. The scope of the invention is defined by the appended claims.

Example 1 design of vaccine platform

The vaccine platform of interferon-target antigen-immunoglobulin Fc (or antibody) structural unit is formed from three structural units, first structural unit is interferon portion, second structural unit is immunoglobulin Fc region (or antibody) and third structural unit is target antigen. In the actual construction, the three structural units can be arranged and combined in any form and the target antigen can be connected with the Th cell helper epitope through the connecting sequence 2. Representative forms are as follows:

FIG. 1 is a schematic diagram of the vaccine platform in a homodimer form, which is combined according to the sequence of interferon-connecting fragment 1-target antigen-immunoglobulin Fc.

FIG. 2 is a schematic representation of the vaccine platform in the form of heterodimers, in terms of interferon-linked fragment 1-IgG1-hole and target antigen-IgG 1-knob combination, respectively.

FIG. 3 is a schematic representation of the vaccine platform in the form of a heterodimer, according to the interferon-linked fragment 1-IgG1-knob and target protein-IgG 1-hole combination.

Next we linked the target antigen to a cell helper epitope by linker fragment 2 and then combined it with two other vaccine platform components, represented by the following forms:

FIG. 4 is a schematic diagram of the vaccine platform in a homodimer form, which is combined according to the sequence of interferon-connecting fragment 1-Th cell helper epitope-connecting fragment 2-target antigen-immunoglobulin Fc.

FIG. 5 is a schematic representation of the vaccine platform in heterodimer form, in terms of interferon-linker 1-IgG1-hole and Th cell helper epitope-linker 2-target antigen-IgG 1-knob combination, respectively.

FIG. 6 is a schematic representation of the vaccine platform in heterodimer form, according to interferon-linker 1-IgG1-knob and Th cell helper epitope-linker 2-target antigen-IgG 1-hole combination, respectively.

Example 2 construction purification and production of vaccine platform

The expression production of the vaccine platform is described by taking the forms of hepatitis B virus Pres1 and coronavirus SARS-CoV-2RBD protein homodimer as examples.

1. Construction of vector, transfection and inducible expression of host cell

1.1, constructing a vaccine structural unit on a vector by taking PEE12.4 as the vector in a molecular cloning mode so as to obtain a plasmid capable of expressing a fusion Protein, then transiently transfecting 293F cells, collecting culture supernatant, and finally purifying a target Protein through a Protein A affinity chromatography column.

Vector construction (taking HBV preS1 antigen as an example)

(1) PEE 12.4-HindIII-Signal peptide 1-Interferon-BsiwI-Pres 1-Bstbi-hIgG1-EcoRI

(2) PEE 12.4-HindIII-Signal peptide 1-Interferon-BsiwI-RBD (SARS-CoV-2) -Bstbi-hIgG1-EcoRI

(3) PEE 12.4-HindIII-Signal peptide 1-Interferon-Bsiwi-PADER-Pres 1-hIgG1-EcoRI

(4) PEE 12.4-HindIII-Signal peptide 1-Interferon

-Bsiwi-PADER-RBD(SARS-CoV-2)-hIgG1-EcoRI

The connection sequence between each fusion protein fragment is

(1) Between interferon and Pres1 is a connecting fragment 1

(2) Between interferon and RBD (SARS-CoV-2) is connecting fragment 1

(3) The connecting sequence between interferon and PADER is a connecting segment 1, the connecting segment between PADER and Pres1 is a connecting segment 2

(4) The connecting sequence between interferon and PADER is connecting fragment 1, the connecting fragment between PADER and RBD (SARS-CoV-2) is connecting fragment 2

1.2, transient transfection to express target protein rapidly:

(1) Cell recovery: freestyle 293F cells at 3X 10 ⁷ The concentration of cells/ml was frozen in CD OptiCHOTM media (10% DMSO contained). After being taken out of liquid nitrogen, the mixture was rapidly melted in a 37 ℃ water bath, and was added to a 15ml centrifuge tube containing 10ml of OptiCHOTM media to be centrifuged at 1,000rpm for 5min. Discarding the supernatant, suspension culturing the cell pellet in 30ml OptiCHOTM media, 37 ℃,8% ₂ And 135rpm. After 4 days, the cells were subjected to scale-up culture at a concentration not exceeding 3X 10 ⁶ cells/ml。

(2) Two days prior to transfection, 293F cells in suspension culture were prepared for transient transfection (200 ml) at a seeding density of 0.6-0.8X 10 ⁶ cells/ml。

(3) After two days, the cell suspensions to be transfected were counted, and the cell density was estimated to be 2.5-3.5X 10 ⁶ cells/ml, followed by centrifugation of the cell suspension at 1,000rpm for 5min and discarding of the supernatant.

(4) The cells were resuspended in 50ml of fresh Freestyle 293 media, centrifuged again at 1,000rpm for 5min and the supernatant discarded.

(5) 293F cells were resuspended in 200ml Freestyle 293 media.

(6) Mu.g of plasmid was diluted with 5ml of Freestyle 293 media and sterilized by filtration using a 0.22. Mu.M filter.

(7) 1.8mg PEI was diluted with 5ml Freestyle 293 media and sterilized by filtration using a 0.22. Mu.M filter. Immediately thereafter, 5ml of the plasmid and 5ml of PEI were mixed and left to stand at room temperature for 5 minutes.

(8) Adding the plasmid/PEI mixture to the cell suspension, standing at 37 ℃,8% ₂ Cultivation in an incubator at 85rpm and simultaneous supplementation of growth factor 50ug/L LONG ^TM R3IGF-1。

(9) After 4 hours, 200ml of EX-CELLTM 293 media and 2mM Glutamine were added, and the rotation speed was adjusted to 135rpm to continue the culture.

(10) After 24 hours, 3.8mM VPA as a cell proliferation inhibitor was added, and after 72 hours, 40ml of medium D was added to continue the culture, and then 6 to 8 days (cell viability: less than 70%) were passed to collect the supernatant for further purification.

1.3 Collection, purification and electrophoretic verification of fusion proteins

2. Protein a was used for purification of the Protein of interest:

(1) Sample preparation: transferring the suspension cell culture solution to a 500ml centrifuge bucket, centrifuging, discarding the precipitate at 8,000rpm,20min, filtering the supernatant with 0.45 μ M filter to remove impurities, and adding NaN to a final concentration of 0.05% ₃ Preventing bacterial contamination during purification.

(2) Assembling a chromatographic column: mixing appropriate amount of Protein A Agarose (calculated by purifying human Fc fusion Protein 20mg per 1ml of Protein A), adding into chromatography column, standing at room temperature for about 10min, opening outlet at bottom after Protein A is layered with 20% ethanol solution, and allowing ethanol solution to slowly flow out under gravity.

(3) The column was washed and equilibrated with 10 column volumes of distilled water and Binding buffer (20 mM sodium phosphate +0.15M NaCl, pH 7.0), respectively.

(4) And (3) sampling by using a constant flow pump, collecting flow-through liquid at the flow rate of 10 times of the column volume per hour, and repeating the sampling for 2 times.

(5) Washing the column by using Binding buffer with the volume more than 10 times of the column volume, washing off impure protein, and washing until effluent liquid is free from protein.

(6) Elution was performed using an Elution Buffer (0.1M Glycine, pH 2.7), the eluate was collected in tubes, 1 tube per 1ml, and the peak was observed using a protein indicator (Bio-Rad protein assay). The collection tubes of the elution peak were mixed and then neutralized with the appropriate amount of 1M Tris at pH 9.0 (pH was adjusted to 6-8, corresponding to a difference of more than 0.5 in isoelectric point of the purified protein).

(7) The protein solution of interest is displaced into the desired buffer using a Zeba desalting spin column or a concentration spin column (care is taken to adjust the buffer pH, avoiding the isoelectric point of the protein). Protein concentration was determined by SDS-PAGE electrophoresis and NanoDrop2000 using BSA as a standard.

(8) After the elution is finished, the column is washed by distilled water with 20 times of column volume in sequence, then the column is washed by 20% ethanol with 10 times of column volume, and finally the gel medium is immersed in the ethanol solution and stored at 4 ℃.

3. The SDS-PAGE electrophoresis identification chart of the protein is shown in FIG. 7.

Example 3 IFN alpha Pres1-Fc, pres1-Fc compared to the simple Pres1 antigen in mice can elicit a stronger immune response.

Materials: c57BL/6 male mice (5-8) were purchased from Experimental animals technology, inc. of Tokyo Wintolidawa, beijing for weeks; goat anti-mouse IgG labeled with horseradish peroxidase (HRP) was purchased from Beijing kang, biotech Co., ltd; 96-well ELISA assay plates were purchased from Corning Costar; ELISA color development was purchased from eBioscience; the microplate reader SPECTRA max PLUS 384 used was purchased from Molecular corporation, USA. The aluminum adjuvant used was purchased from SIGMA.

The method comprises the following steps:

(1) The mice were immunized with the Pres1 fusion protein, and the mice were immunized subcutaneously by mixing 80pmol IFN-Pres1-Fc or 80pmol Pres1-Fc or Pres1 protein with aluminum adjuvant. Serum from mice was collected by orbital bleeds at the indicated time points for antibody detection.

(2) The antibody produced by IFN alpha-Pres 1-Fc has wide neutralizing effect on HBV viruses with different genotypes. 5-week-old male C57BL/6 is infected with AAV-HBV1.3 (wherein HBV genotypes are B, C and D) 1x10 by means of tail vein ¹¹ vg virus, mice with persistent and stable expression of HBV antigen were screened after 6 weeks for experiments. The selected mice (4 mice/group) were injected with 200. Mu.l/mouse of serum from IFN α -Pres1-Fc immunized mice by intravenous injection. After 12 hours, mouse serum was collected and the changes of Pres1 antigen in mice before and after injection of antiserum were examined by means of Elisa.

(3) ELISA was used to detect anti-Pres1 specific antibodies in serum. Pres1 (2. Mu.g/ml) coating solution was added to an Elisa plate (Corning 9018) in a system of 50. Mu.l per well, and coated overnight at 4 ℃. Wash once with PBS, 260. Mu.l per well. 5% blocking solution (5% FBS) blocked for two hours at 37 ℃. Serum samples were diluted with PBS (1: 10, 1: 1)100, 1: 1000, 1: 10000), 50ul per well was added to a closed Elisa plate and incubated at 37 ℃ for 1 hour. PBST was washed 5 times with 260. Mu.l each, and 50. Mu.l enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1: 5000diluted by PBS) was added to each well and incubated at 37 ℃ for 1 hour. Washing with PBST for five times, wherein 260 mu l of PBST is added, 100 mu l/hole of TMB is added, incubation is carried out at room temperature in a dark place, and the substrate is waited for color development; add 50. Mu.l of stop solution (2 NH) per well ₂ SO ₄ ) The color development was stopped and the plate was read with a microplate reader, OD450-630.

As a result: free Pres1 is weaker in immunogenicity, and the immunogenicity is greatly improved when IFN alpha and Fc parts are added on the basis of Pres1 to form IFN alpha-Pres 1-Fc fusion protein, as shown in FIG. 8 (a). And the antibody caused by IFN alpha-Pres 1-Fc can produce wide neutralization effect on different HBV genotype viruses, as shown in figure 8 (b).

Example 4 IFN alpha Pres1-Fc can be used as hepatitis B prophylactic vaccine

Materials: c57BL/6 (6-8) Zhou male mice from Beijing Wei Tongli Hua biotechnology, inc., HBsAg detection kit from Shanghai Kehua bioengineering, inc. AAV-HBV1.3 virus was purchased from Peizhou Paizhii Biotechnology, inc. Other experimental materials were the same as in example 3.

The method comprises the following steps:

(1) Mice are inoculated with 80pmol of different forms of Pres1 vaccines including simple Pres1, pes1-Fc and IFN alpha-Pres 1-Fc protein in a subcutaneous immunization manner, and at 28 days of immunization, mouse serum is collected and infected with 1x10 ¹¹ vgAAV-HBV 1.3 virus, then collecting mouse serum weekly to detect anti-Pres1 antibody, HBsAg and Pres1 antigen in serum, and continuously detecting for four weeks. Peripheral HBV-DNA levels in mice were measured at week three.

(2) ELISA detects Pres 1-specific antigen in serum. Antigen coating: the coating solution of Pres1 antibody XY007 (4. Mu.g/ml) was applied to an Elisa plate (Corning 9018) in a volume of 50. Mu.l per well, and was coated overnight at 4 ℃. Wash once with PBS, 260. Mu.l per well. Blocking with 5% blocking solution (5% FBS) for two hours at 37 ℃. Serum samples (1: 10, 1: 100) were diluted in PBS and 50. Mu.l were added to the well-blocked Elisa plates, two for each dilution setMultiple wells were incubated at 37 ℃ for 1 hour. PBST was washed 5 times with 260. Mu.l each, 50. Mu.l of enzyme conjugate (from Kowa HBsAg detection Kit) was added to each well and incubated at 37 ℃ for 1 hour. Washing five times with PBST, 260 mul each time, adding 100 mul/hole of TMB as substrate, incubating at room temperature in dark place, and waiting for substrate color development; add 50. Mu.l of stop solution (2 NH) per well ₂ SO ₄ ) The color development is stopped, and the plate is read by a microplate reader, OD450-630.

As a result: the IFN alpha-Pres 1-Fc group of mice before inoculation of virus can produce high levels of Pres1 antibody and antibody in the course of virus infection continuously maintained at high levels as shown in figure 9 (a). Compared with the group without protein immunization, the IFN-Pres1-Fc vaccine immunization can remarkably prevent HBV infection, the anti-preS1 antibody generated after immunization can quickly and completely eliminate the preS1 antigen in serum in figure 9 (b), and most of mice receiving virus infection in the IFN-Pres1-Fc immunization group are shown as peripheral HBsAg negative figure 9 (c, d). The above experimental results indicate that IFN-Pres1-Fc as a vaccine can effectively prevent HBV infection, as shown in FIG. 9.

Example 5 IFN alpha Pres1-Fc as a chronic infection of B therapeutic vaccine

Materials: c57BL/6 male mice (4 weeks) were purchased from Biotechnology Ltd, viton, beijing. AAV-HBV1.3 was purchased from Peizhou Pachy Biotechnology, inc. The HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., ltd, and the other experimental materials were the same as in example 4.

The method comprises the following steps:

(1) Screening of HBV Carrier mice, 4 week old HBV C57BL/6 mice injected by tail vein 1X10 ¹¹ vg AAV-HBV1.3 virus, 1-6 weeks through detecting HBV antigen HBsAg, screening out

HBsAg stably expressed mice, as HBV Carrier mice for experiment.

(2) The selected mice were immunized three times in total every two weeks by injecting 80pmol of different forms of Pres1 protein subcutaneously. Mouse serum was collected at 14 days of immunization, and then collected once a week, and the levels of anti-Pres1 antibody, HBsAg, and Pres1 antigen in the mouse serum were measured by ELISA. The HBV-DNA content in the peripheral blood of the mice was measured after the last blood draw.

As a result: we examined the changes of Pres1 antigen in the serum of IFN-Pres1-Fc vaccine immunized Carrier mice, and Pres1 antibody and HBsAg in the serum. The results show that, after IFN alpha-Pres 1-Fc vaccine immunization, the mouse high level anti-Pres1 antibody production is shown in figure 10 (a), and the serum Pres1 antigen can be completely eliminated, as shown in figure 10 (b). Meanwhile, HBsAg in serum is reduced to some extent as shown in FIG. 10 (c), while the untreated control group and the Pres1 vaccine immunization group alone have no therapeutic effect as shown in FIG. 10.

Example 6T cell helper epitopes enhance the antibody response of IFN alpha Pres1-Fc vaccine

Materials: same as example 3

The method comprises the following steps:

(1) The mice were immunized with Pres1 fusion protein, and 80pmol of IFN-Pan-Pres1-Fc containing a Pan epitope or 80pmol of IFN-Pan-Pres1-Fc, pres1 protein were subcutaneously immunized with the mice. Sera from mice were collected by orbital bleeds at the indicated time points for antibody detection.

(2) ELISA was performed to detect anti-Pres1 specific antibody in serum as in example 3.

As a result: compared with the fusion protein vaccine such as IFN-preS1-Fc, the IFN-Pan-preS1-Fc can obviously enhance the immunogenicity of antigen molecules and can cause the generation of broad-spectrum neutralizing antibodies. C57/BL6 (n = 8/group) mice were inoculated with hepatitis b Pres1, pres1-Fc, IFN α -Pres1-Fc proteins without aluminum adjuvant by subcutaneous immunization, and the level of Pres 1-specific antibodies in serum was measured at the indicated time using the method of Elisa.

Example 7 IFN alpha Pan Pres1-Fc as chronic infection of B therapeutic vaccine

The method comprises the following steps:

(1) Screening of HBV Carrier mice4 week old HBV C57BL/6 mice were injected 1X10 by tail vein ¹¹ vg AAV-HBV1.3 virus, HBsAg is detected in 1-6 weeks, and HBsAg stably expressed mice are selected and used as HBV Carrier mice for experiment.

(2) The selected mice were immunized three times in total every two weeks by injecting 80pmol of different forms of Pres1 protein subcutaneously. Mouse serum was collected at 14 days of immunization, and then taken once a week, and the levels of anti-Pres1 antibody, HBsAG, pres1 antigen in the mouse serum were measured by ELISA. The HBV-DNA content in the peripheral blood of the mice was measured after the last blood draw.

As a result: we examined the IFN-Pan-Pres1-Fc vaccine immune Carrier mice, its serum PreS1 antigen, and serum Pres1 antibody and HBsAg changes. The results show that, after IFN-Pan-Pres1-Fc vaccine immunization, mice produced high levels of anti-Pres1 antibody as shown in FIG. 12 (a). And the pre S1 antigen in the serum can be completely eliminated as shown in figure 12 (b), and the HBsAg in the serum is reduced to a certain degree 12 (c), while the untreated control group and the Pres1 vaccine immunization group alone have no treatment effect. And HBV DNA was also significantly decreased in the IFN α -Pan-Pres1-Fc immunized group as shown in FIG. 12 (d).

Example 8 IFN alpha Pan Pres1-Fc in combination with HBsAg commercial vaccine, breaking the immune tolerance against HBsAg, induce HBsAg-HBsAb serologic switch.

Materials: c57BL/6 male mice (4 weeks) were purchased from Biotechnology, inc., viton, beijing. AAV-HBV1.3 was purchased from Guangzhou Peizhen Biotechnology, inc. The HBsAg detection kit is purchased from Shanghai Kehua biotechnology, inc., and the Anti-HBsAg kit is purchased from Beijing Wantai biopharmaceutical, inc. Commercial HBsAg vaccines are purchased from eimeria hanxin vaccine (da lian) limited. The other experimental materials were the same as in example 7.

The method comprises the following steps:

(1) Screening of HBV Carrier mice, 4 week old HBV C57BL/6 mice injected by tail vein 1X10 ¹¹ vg AAV-HBV1.3 virus, 1-6 weeks through detecting HBV antigen HBsAg, screening HBsAg stabilizationThe expressed mice were used as HBV Carrier mice for experiments.

(2) The screened HBV Carrier mice were immunized with 80pmol IFN alpha-pan-Pres 1-Fc, and simultaneously with 2. Mu.g of commercial HBsAg vaccine, were immunized twice consecutively with an interval of 14 days. Mouse sera were collected 14 days after the first immunization, and then weekly, and changes in anti-Pres1, anti-HBsAg, HBsAg in the sera were examined. And detecting the level of HBV-DNA in the serum at the time of the last mouse serum collection.

As a result: we found that the combination of IFN alpha-Pan-Pres 1-Fc and commercial HBsAg as a strategy for treating chronic hepatitis B can eventually break the HBsAg tolerance. The immune response in HBV-tolerant mice was able to completely eliminate the preS1 antigen in serum FIG. 13 (a), and high concentrations of preS1 antibody 13 (c) were present in serum. Exciting, the IFN-Pan-Pres1-Fc vaccine simultaneously effectively cleared HBsAg in serum, and simultaneously induced partial serological HBsAb transformation in FIGS. 13 (b) and 4 (d), which is clinically recognized as a key indicator of HBV cure. In addition, we have detected the expression level of HBV-related DNA in peripheral blood by the method of fluorescence quantitative PCR (real-time PCR), respectively, and the results show that the immunization mode of combining IFN alpha-Pan-Pres 1-Fc with commercial HBsAg can finally reduce the level of peripheral HBV DNA relative to the control group, and through the results, we have invented the vaccine strategy for treating chronic hepatitis B by combining IFN alpha-Pan-Pres 1-Fc with commercial HBsAg vaccine

Example 9 IFN alpha-RBD (SARS-CoV 2) -Fc elicits a stronger antibody response than free SARS-Cov 2RBD protein

Materials: balb/c Male and female mice (6-8 weeks) were purchased from Beijing Wittingerli laboratory animal technology, inc., and the SARS-CoV-2RBD protein used was purchased from Beijing Kongzhongji Biotechnology, inc. 293-hACE2 cells were provided by Zhang professor (third national Hospital, shenzhen).Luciferase ReporteThe r detection kit was purchased from promega corporation.

Other experimental materials were the same as in example 3.

The method comprises the following steps:

(1) IFN alpha-RBD (SARS-Cov-2) -Fc fusion protein immune mice, 10u g IFN alpha-RBD-Fc, RBD-Fc or 10u g RBD protein and aluminum adjuvant after mixing subcutaneous immune mice. Serum from mice was collected by orbital bleeds at 28 days of immunization for detection of neocoronal specific antibodies.

(2) Serum SARS-cov 2RBD antibodies were detected. Antigen coating: RBD (1.5. Mu.g/ml) coating solution was added to an Elisa plate (Corning 9018) in a system of 100. Mu.l per well, and coated overnight at 4 ℃. Wash once with PBS, 260. Mu.l per well. Blocking was performed at 37 ℃ for two hours using 100. Mu.l of 5% blocking solution (5% FBS). Serum samples (1: 10, 1: 100, 1: 1000, 1: 10000, 1: 100000.. ANG.) were diluted with PBS and 100. Mu.l per well were incubated in a closed Elisa plate for 1 hour at 37 ℃. PBST was washed 5 times with 260. Mu.l each, and 100. Mu.l of enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1: 5000diluted by PBS) was added to each well and incubated at 37 ℃ for 1 hour. The column was washed five times with PBST, 260. Mu.l each time, substrate TMB was added at 100. Mu.l/well, incubated for 15 minutes at room temperature in the dark, and the substrate was allowed to develop. Add 50. Mu.l of stop solution (2 NH) per well ₂ SO ₄ ) The color development is stopped, and the plate is read by a microplate reader, OD450-630. And (3) a titer calculation method, namely selecting the maximum dilution factor with positive result, and multiplying the OD value/Cutoff value (0.1) corresponding to the dilution factor by the (X) dilution factor to obtain the value which is the titer of the antibody corresponding to the serum.

(3) SARS-CoV-2S protein pseudovirus in vitro neutralization experiment. Antiserum was diluted 1: 3 into 96-well plates, 50ul pseudoviral particles with luciferase spike protein were added to the wells, the virus-antibody mixture was left at 37 ℃ for 1 hour, and 10^4 cells per well of 293-hACE2 were added to 96-well plates. The 96-well plate was placed in a 37 ℃ cell incubator, and the activity of luciferase was measured after 48 hours.

As a result: free new crown RBD has weaker immunogenicity, and when IFN alpha and Fc parts are added on the basis of the polypeptide protein region of the new crown RBD to form IFN alpha-RBD-Fc fusion protein, the immunogenicity is greatly improved, as shown in figure 14. And the antibody caused by IFN alpha-RBD-Fc can block the infection of cells by the pseudovirus of SARS-CoV-2S protein in vitro as shown in FIG. 15.

Example 10 detection of antiserum RBD-specific antibodies generated by immunization with FN α -Pan-RBD (original strain) -Fc and IFN α -RBD (SARS-CoV-2 south African mutant) -Fc.

Materials: balb/c male and female mice (6-8 weeks) were purchased from Beijing Wintoli Kaishu laboratory animal technology, inc., and the used SARS-CoV-2 original strain RBD protein was purchased from Beijing Kezhongzheng Kanjing Biotechnology, inc. The SARS-CoV-2 south Africa mutant strain RBD protein was purchased from Beijing Yiqian Shenzhou science and technology Co.

Other experimental materials were the same as in example 3.

The method comprises the following steps:

(1) The IFN alpha-Pan-RBD (original strain) -Fc and IFN alpha-RBD (SARS-CoV-2 south Africa mutant) -Fc protein construction and expression method are the same as in example 2.

(2) IFN alpha-Pan-RBD (original strain) -Fc and IFN alpha-Pan-RBD (SARS-CoV-2 south African mutant) -Fc fusion protein immune mice, IFN alpha-Pan-RBD (original strain) -Fc or IFN alpha-Pan-RBD (SARS-CoV-2 south African mutant) -Fc protein and aluminum adjuvant mixed after subcutaneous immune mice. Serum from mice was collected by orbital bleeds at day 14 of immunization for detection of neocorona-specific antibodies.

(3) The antibody response was analyzed by ELISA as in example 9.

As a result: the SDS-PAGE results showed correct band size for IFN α -Pan-RBD (SARS-CoV-2 original strain) -Fc, indicating that the mutant neocoronavirus IFN α -RBD (SARS-CoV-2 original strain) -Fc vaccine protein was successfully constructed, expressed and purified (FIG. 16 a), and the SDS-PAGE results showed correct band size for IFN α -Pan-RBD (SARS-CoV-2 south African mutant) -Fc, indicating that the mutant neocoronavirus IFN α -RBD (SARS-CoV-2 south African mutant) -Fc vaccine protein was successfully constructed, expressed and purified (FIG. 16 b). The ELISA results showed that IFN α -Panan-RBD (original strain) -Fc immunized mice, and the induced antibodies could bind to the original strain of new coronavirus RBD protein in IFN α -Pan-RBD (SARS-CoV-2 south African mutant) -Fc immunized mice, and the binding ability of the induced antibodies to the original strain RBD was not significantly different (FIG. 16 c). Meanwhile, the ELISA results for the south African mutant RBD also show that IFN alpha-Panan-RBD (original strain) -Fc immune mice, and IFN alpha-Pan-RBD (SARS-CoV-2 south African mutant) -Fc immune mice can induce the generation of antibodies which can be combined with the south African mutant RBD with the same combination ability (FIG. 16 d).

Example 11.

Materials: c57BL/6 female mice (6-8 weeks) were purchased from Biotechnology Ltd, viton, beijing. SARS-CoV-2RBD protein for ELISA was purchased from Beijing Ke Jin Zhongji Biotechnology Co., ltd; the Mouse IFN alpha-RBD-Fc, mouse IFN alpha-Pan-RBD-Fc, human IFN alpha-RBD-Fc and Human IFN alpha-Pan-RBD-Fc proteins for immunization were all produced in the laboratory, and other experimental materials were the same as in example 3.

The method comprises the following steps:

(1) The fusion protein design, plasmid construction and protein purification methods are as described in examples 1 and 2.

(2) Mice were immunized with vaccine proteins. Mu.g of Mouse IFN alpha-RBD-Fc, mouse IFN alpha-Pan-RBD-Fc or 10 mu g of Human IFN alpha-RBD-Fc and Human IFN alpha-Pan-RBD-Fc vaccine protein are respectively mixed with 20 mu g of aluminum adjuvant overnight, then the mice are inoculated by means of muscle immunization, and one boosting immunization is carried out 14 days after the primary inoculation. Mouse sera were collected at 7, 14, and 28 days after immunization, and the level of RBD-specific antibodies in the mouse sera was measured by ELISA.

(3) Serum SARS-cov 2RBD antibodies were detected. Antigen coating: RBD (1.5. Mu.g/ml) coating solution was added to an Elisa plate (Corning 9018) in a system of 100. Mu.l per well, and coated overnight at 4 ℃. Wash once with PBS, 260. Mu.l per well. Blocking was performed at 37 ℃ for two hours using 100. Mu.l of 5% blocking solution (5% FBS). Serum samples (1: 10, 1: 100, 1: 1000, 1: 10000, 1: 100000.. ANG.) were diluted with PBS and 100. Mu.l per well were incubated in a closed Elisa plate for 1 hour at 37 ℃. PBST was washed 5 times with 260. Mu.l each, and 100. Mu.l of enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1: 5000diluted by PBS) was added to each well and incubated at 37 ℃ for 1 hour. The column was washed five times with PBST, 260. Mu.l each time, substrate TMB was added at 100. Mu.l/well, incubated for 15 minutes at room temperature in the dark, and the substrate was allowed to develop. Add 50. Mu.l of stop solution (2 NH) per well ₂ SO ₄ ) The color development was stopped and the plate was read with a microplate reader, OD450-630. The titer calculation method comprises selecting the maximum dilution factor with positive result, and multiplying the OD value/Cutoff value (0.1) corresponding to the dilution factor by the (X) dilution factor to obtainThe value of (b) is the corresponding antibody titer of the serum.

As a result:

as shown in fig. 17, after the protein was purified by expression, SDS-PAGE results indicated that the protein size was as expected and exhibited a single band at the target position.

As shown in FIG. 18, the addition of the Pan (Pan DR-binding epitope) CD 4T cell helper epitope enhanced the immunity of Mouse IFN α -RBD-Fc and Human IFN α -RBD-Fc. The experimental results show that addition of the monse IFN α -Pan-RBD-Fc compared to the monse IFN α -RBD-Fc, and the Human IFN α -Pan-RBD-Fc compared to the Human IFN α -RBD-Fc, resulted in higher production of RBD-specific antibodies, whether on day 7 or day 14, 28 after immunization of the vaccine proteins.

Example 12

Materials: c57BL/6 female mice (6-8 weeks) were purchased from Beijing Wittingle Biotechnology, inc.; SARS-CoV-2RBD protein for ELISA was purchased from Beijing Ke Jin Zhongji Biotechnology Ltd. The Human IFN alpha-RBD-Fc and Human IFN alpha-Pan-RBD-Fc proteins for immunization were produced in the laboratory. Other experimental materials were the same as in example 3.

The method comprises the following steps:

(1) Human IFN alpha-RBD-Fc and Human IFN alpha-Pan-RBD-Fc protein. Mu.g of Human IFN α -RBD-Fc or Human IFN α -Pan-RBD-Fc protein was mixed with aluminum adjuvant overnight as a vaccine sample containing aluminum adjuvant, and another set of 10. Mu.g of Human IFN α -RBD-Fc or Human IFN α -Pan-RBD-Fc protein was diluted with PBS as a vaccine sample without adjuvant. Mice were vaccinated intramuscularly with 10 μ g of Human IFN α -RBD-Fc, human IFN α -Pan-RBD-Fc protein, in the presence or absence of aluminum adjuvant, and a booster immunization was performed 14 days after vaccination. Mouse sera were collected at 7, 14, and 28 days after immunization, respectively, and the RBD-specific antibody level in the mouse sera was measured by ELISA.

(2) Serum SARS-cov 2RBD antibodies were detected. Antigen coating: RBD (1.5. Mu.g/ml) coating solution was added to an Elisa plate (Corning 9018) in a system of 100. Mu.l per well, and coated overnight at 4 ℃. Wash once with PBS, 260. Mu.l per well. With 5% ofBlocking solution (5% FBS) 100. Mu.l, for two hours at 37 ℃. Serum samples (1: 10, 1: 100, 1: 1000, 1: 10000, 1: 100000.) were diluted with PBS and incubated for 1 hour at 37 ℃ in 100. Mu.l per well in a closed Elisa plate. PBST was washed 5 times with 260. Mu.l each, and 100. Mu.l enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1: 5000diluted by PBS) was added to each well and incubated at 37 ℃ for 1 hour. The column was washed five times with PBST, 260. Mu.l each time, substrate TMB was added at 100. Mu.l/well, incubated for 15 minutes at room temperature in the dark, and the substrate was allowed to develop. Add 50. Mu.l of stop solution (2 NH) per well ₂ 8O ₄ ) The color development was stopped and the plate was read with a microplate reader, OD450-630. And (3) a titer calculation method, namely selecting the maximum dilution factor with positive result, and multiplying the OD value/Cutoff value (0.1) corresponding to the dilution factor by the (X) dilution factor to obtain the value which is the titer of the antibody corresponding to the serum.

As a result:

as shown in FIG. 19, the application of aluminum adjuvant can enhance the immunogenicity of Human IFN alpha-RBD-Fc and Human IFN alpha-Pan-RBD-Fc proteins. Although the non-adjuvant Human IFN alpha-RBD-Fc and Human IFN alpha-Pan-RBD-Fc vaccines can generate high titer antibody responses, the aluminum adjuvant-assisted Human IFN alpha-RBD-Fc and Human IFN alpha-Pan-RBD-Fc proteins can further improve the RBD specific antibody response level on

days

7, 14 and 28 after inoculation compared with the non-adjuvant-assisted group.

Example 13.

Materials:

the experimental animals are purchased from Beijing Wittiulihua experimental animals Co., ltd, and the animals are 6-8 weeks C57BL/6 mice; animal certification numbering: no.110011200106828974; RBD protein for immunization was purchased from Beijing Ke Ji Zhong Ji Biotechnology Co., ltd; RBD-Fc, IFN alpha-RBD-Fc and IFN-pan-RBD-Fc protein are produced by the laboratory; all adjuvants were purchased from SERVA, germany; horse radish peroxidation enzyme (HRP) labeled goat anti-mouse IgG is purchased from Beijing kang, biotechnology GmbH; 96-well ELISA assay plates were purchased from Corning Costar; ELISA color development solutions were purchased from eBioscience; the microplate reader SPECTRA max PLUS 384 used was purchased from Molecular corporation, USA; tissue homogenate disruption apparatus was purchased from beijing hao nuo s technologies ltd.

The method comprises the following steps:

the mice are divided into 5 groups in 6-8 weeks, each group contains 10 mice, and 10 mu g of IFN alpha-pan-RBD-Fc or RBD, RBD-Fc and IFN alpha-RBD-Fc protein with the same molar number is immunized by nasal drip immunization, and the nasal drip dose is 10 mu L per mouse. Mice were immunized on days 0 and 14 using two immunization programs. Collecting mouse serum on 7, 14, 21, 28, 35 and 42 days after immunization, and detecting the content of SARS-CoV-2RBD specific antibody in each group of serum by ELISA method; serum from 28 days was taken and subjected to the SARS-CoV-2 pseudovirus in vitro neutralization assay.

As a result:

as shown in FIG. 20, the RBD, RBD-Fc protein can cause a certain degree of antibody reaction by two nasal drops, the serum IgG and IgA levels induced by the IFN alpha-pan-RBD-Fc after two nasal drops are obviously higher than those induced by the IFN alpha-pan-RBD-Fc protein at the same time point, and the results of the pseudovirus neutralization experiment show that the IFN-RBD-Fc protein can induce higher level of neutralizing antibody production compared with the RBD and RBD-Fc immune group.

Example 14.

Materials:

same as example 10

The method comprises the following steps:

the mice were divided into 4 groups of 5 mice each at 6-8 weeks, and 10. Mu.g of IFN α -pan-RBD-Fc or the same number of moles of RBD, RBD-Fc, IFN α -RBD-Fc protein was immunized by nasal drip immunization at a dose of 10. Mu.L per mouse. Mice were immunized on days 0 and 14 using two immunization programs. Collecting mouse nasal mucosa supernatant and lung lavage liquid at 28 days after immunization, detecting SARS-CoV-2RBD specific antibody content in each group of serum by ELISA method, and detecting serum and new corona pseudovirus neutralization experiment of nasal mucosa supernatant by SARS-CoV-2 pseudovirus neutralization experiment.

Obtaining nasal mucosa supernatant and alveolar lavage fluid of an immune experimental animal mouse: after the mice die in dormancy, the nasal mucosa of the mice is taken and crushed by a tissue homogenate crusher. And (3) centrifuging the homogenized liquid at 13000rpm for 10 minutes at a high speed, and taking the supernatant as nasal mucosa supernatant (NMDS). In the lung of the mouse, about 0.8ml of HBSS +100 mu MEDTA is sucked by a 1ml syringe, the mouse is inserted into an air tube, after the mouse is gently and repeatedly blown and sucked for three times, the liquid is sucked out and collected into a centrifugal tube, and the step is repeated for three times, so that about 2ml of lung lavage liquid can be finally obtained. And centrifuging 500g of the mouse lung lavage fluid for 5 minutes to obtain a supernatant, namely the mouse lung lavage fluid (BALF), precipitating lymphocytes in the mouse lung, and performing further analysis.

As a result:

as shown in FIG. 21, IFN α -pan-RBD-Fc protein compared to RBD and RBD-Fc proteins, two nasal drops of immunization elicited strong nasal mucosal local IgG antibody responses and IgA mucosal immunization. The reaction strength of the IFN alpha-pan-RBD-Fc protein is stronger than that of the RBD and RBD-Fc groups. The results of the pseudovirus neutralization experiments show that IFN alpha-pan-RBD-Fc protein immunization groups can induce higher titer neutralizing antibody production at nasal mucosa.

As shown in FIG. 22, IFN α -pan-RBD-Fc fusion protein two nasal drops immunized C57BL/6 mice also induced strong secretion of IgG and IgA antibodies in local lung tissues. The results of the pseudovirus neutralization experiments show that IFN alpha-pan-RBD-Fc induces production of neutralizing antibodies with higher titer compared with RBD and RBD-Fc.

Example 15.

Her2 belongs to the type I transmembrane growth factor receptor Her family and 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 dimerizes and transphosphorylates its intracellular domain, and the phosphorylated tyrosine residue can bind to a variety of intracellular signaling molecules, activating downstream signaling pathways, and regulating gene transcription. The regulated genes are mostly involved in processes such as cell proliferation, survival, differentiation, angiogenesis, invasion and metastasis. The Her2 protein has a large extracellular segment with more than 600 amino acids, and can be divided into four structural domains, namely structural domains I, II, III and IV. Currently approved Trastuzumab binds primarily to domain IV, pertuzumab binds primarily to domain II, and the polypeptide vaccine E75 that is undergoing clinical trials targets domain III. Indicating that the different domains have some important sites, which may mediate antitumor effects. In order to research the application of the vaccine platform in tumor prevention and treatment, the patent selects a tumor antigen Her2 as a target spot, constructs IFN-Her2-Fc and IFN-Pan-Her2-Fc to construct a fusion protein vaccine, and analyzes in-vivo anti-tumor activity and vaccine immunocompetence.

Materials and methods:

materials:

BALB/c female mice (6-8 weeks) were purchased from Biotech, inc., viton, beijing; TUBO cells are derived from TCGA; the other materials were the same as in example 3.

The method comprises the following steps:

(1) Fusion protein design, plasmid construction and protein purification procedures are shown in examples 1 and 2.

Expression plasmids (denoted IFN alpha-3-Fc, IFN alpha-pan-4-Fc and IFN alpha-4-Fc, respectively) were first constructed against domains III and IV of the extracellular domain of mouse Her2, and the relevant proteins were expressed and purified in the human 293F cell line. The size and purity of the protein was identified by SDS-PAGE and Coomassie blue staining.

(2) Analysis of IFN alpha-3-Fc and IFN alpha-pan-3-Fc direct antitumor Activity

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 IFN alpha in the protein is detected by using TUBO tumor. Construction of TUBO Breast cancer model mice, 5 x10 ⁵ TUBO cells are inoculated to BALB/C mice subcutaneously, and the tumor size is 50-80mm ³ Treatment was given once a week for a total of 3 treatments. The dose of IFN alpha-3-Fc was 10 ug/mouse, other drugs were administered equimolar, cpG as adjuvant. Tumor size was measured and tumor growth curves were plotted.

(3) Analysis of the enhancement of immunogenicity of IFN alpha and Pan to Her2 vaccines

BALB/C female mice at 6-8 weeks were subcutaneously vaccinated 3 times 1 time per week with the HER2 domain V fusion protein vaccine 4-Fc, IFN alpha-4-Fc and IFN alpha-pan-4-Fc without adjuvant. The immunization dose is IFN alpha-4-Fc 10 ug/mouse, and other proteins are inoculated in an equimolar way. Venous blood was removed 14 days after immunization at 21 days and antibody levels of Her2 specific IgG were measured by ELISA.

As a result:

(1) As shown in FIG. 23, the Her2 fusion protein basically meets the expected size and reaches the experimental requirement in purity. IFN alpha-3-Fc (62.6 kDa), IFN alpha-pan-3-Fc (63.9 kDa), IFN alpha-pan-4-Fc (74.9 kDa) and IFN alpha-4-Fc (73.6 kDa). Under non-deformation conditions, the protein is in a dimer state and conforms to the characteristic of automatic dimerization of Fc fragments. (2) As shown in FIG. 24, the intra-tumor injection of Her2 fusion proteins IFN α -pan-3-Fc and IFN α -3-Fc significantly inhibited the growth of TUBO tumors compared to the control group, and the control effect was comparable to the IFN α -Fc group. The protein vaccine shows that the activity of the IFN alpha in the protein vaccine is good, the factors such as steric hindrance and the like do not appear to influence the activity of the IFN alpha, and the protein vaccine can be used for further researching the efficacy and mechanism of the protein vaccine in antitumor immunity.

(3) As shown in FIG. 25, 4-Fc, IFN α -4-Fc and IFN α -pan-4-Fc induced significant Her 2-specific IgG antibody responses 14 days and 21 days after Her2 fusion protein vaccine immunization compared to the control group; IFN alpha-4-Fc and IFN alpha-pan-4-Fc induced antibody titers tended to increase compared to 4-Fc. Furthermore, IFN α -pan-4-Fc induced antibody titers at 21 days post-immunization were significantly higher than in the 4-Fc group. It is demonstrated that the addition of IFN alpha and Pan contributes to increase 4-Fc immunogenicity, inducing a more robust antigen-specific antibody response, and therefore IFN-Pan-HER2-Fc and IFN-Pan-HER2-Fc are potential effective tumor vaccines against Her2 positive tumors.

Example 16.

Materials: BALB/c female mice (6-8 weeks) were purchased from Biotech, inc., viton, beijing; HA1 (A/PR 8) protein for ELISA was purchased from Beijing-Qiao Shenzhou Biotechnology, inc.; HA1 protein (A/PR 8) for immunization is purchased from Beijing-Qiao Shenzhou biotechnology limited, and IFN alpha-HA 1-Fc is produced in the laboratory; H1N1 (A/PR 8) influenza viruses used to infect mice were produced by the laboratory; other experimental materials were the same as in example three.

The method comprises the following steps:

(1) The design, plasmid construction and protein purification of the IFN alpha-HA 1-Fc protein are as described in examples 1 and 2.

(2) HA1, IFN alpha HA1-Fc protein immune mice. After 10. Mu.g of IFN α -HA1-Fc or the same molar amount of HA1 protein, respectively, were mixed with 20. Mu.g of aluminum adjuvant overnight, the mice were vaccinated by means of muscle immunization and a booster immunization was performed 14 days after the initial vaccination. Mouse sera were collected at day 28 after immunization and assayed for HA 1-specific antibody levels by ELISA.

(3) Serum HA1 antibodies were detected. Antigen coating: HA1 (2. Mu.g/ml) coating solution was added to an Elisa plate (Corning 9018) in a system of 100ul per well and coated overnight at 4 ℃. Wash once with PBS, 260. Mu.l per well. Blocking was performed at 37 ℃ for two hours using 100. Mu.l of 5% blocking solution (5% FBS). Serum samples (1: 10, 1: 100, 1: 1000, 1: 10000, 1: 100000.. ANG.) were diluted with PBS and 100. Mu.l per well were incubated in a closed Elisa plate for 1 hour at 37 ℃. PBST was washed 5 times, each at 260. Mu.l, and 100. Mu.l of enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1: 5000diluted by PBS) was added to each well and incubated at 37 ℃ for 1 hour. The column was washed five times with PBST, 260. Mu.l each time, substrate TMB was added at 100. Mu.l/well, incubated for 15 minutes at room temperature in the dark, and the substrate was allowed to develop. Add 50. Mu.l of stop solution (2 NH) per well ₂ SO ₄ ) The color development is stopped, and the plate is read by a microplate reader, OD450-630. And (3) a titer calculation method, namely selecting the maximum dilution factor with positive result, and multiplying the OD value/Cutoff value (0.1) corresponding to the dilution factor by the (X) dilution factor to obtain the value which is the titer of the antibody corresponding to the serum.

(4) Mice were anesthetized 42 days after immunization and infected with 1000PFU A/PR8 influenza virus by nasal drip, and the mice were observed every two days and weighed on the third day after infection.

As a result:

as shown in FIG. 26, the size and purity of the protein were determined by SDS-PAGE after the protein was purified by expression, and the result showed that a single band was exhibited at the size of the band of interest. As shown in fig. 27, IFN α -HA1-Fc induced higher titers of HA 1-specific antibodies than the HA1 protein (fig. 27 a), suggesting that the vaccine platform may enhance the immunogenicity of the HA1 protein. The body weight of the mice after being attacked by the virus changes obviously, but the body weight of the mice in the IFN alpha-HA 1-Fc immune group rises rapidly compared with that in the PBS group and the HA1 protein immune group, which indicates that the IFN alpha-HA 1-Fc vaccine shows good protection to influenza infection (figure 27 b).

Example 17

Materials and methods:

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 as shown in examples 1 and 2.

As a result:

as shown in FIG. 28, after 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 determined by SDS-PAGE, which indicated that the positions of the target bands were correct.

Example 18

Compared with free EBV-gp350 protein, IFNa-Pan-EBV gp350-Fc can cause stronger humoral immune response

Materials: c57BL/6 female mice (6-8 weeks) were purchased from Beijing Wittingle Biotechnology, inc.; EBV-gp350 protein for ELISA was purchased from Baiying Biotech, inc., tanzhou; the EBV-gp350 protein for immunization is purchased from Baiying Biotechnology GmbH, thai, and IFN alpha-Pan-EBV gp350-Fc is produced in the laboratory; other experimental materials were the same as in example three.

The method comprises the following steps:

(1) The EBV gp350 and IFN alpha-Pan-EBV gp350-Fc proteins immunize mice, 10 mu g of IFN alpha-Pan-EBV gp350-Fc or the same molar amount of EBV-gp350 protein is respectively mixed with 20 mu g of aluminum adjuvant overnight, then the mice are inoculated by means of muscle immunization, and one boosting immunization is carried out 14 days after the primary inoculation. Mouse sera were collected at day 14 and 28 after the primary immunization and assayed for levels of EBV-gp 350-specific antibodies by ELISA.

(2) And detecting the serum EBV-gp350 antibody. Antigen coating: EBV-gp350 (1. Mu.g/ml) coating solution was added to an ELISA plate (Corning 9018) in a volume of 100. Mu.l per well and coated overnight at 4 ℃. Wash once with PBS, 260. Mu.l per well. Blocking was performed with 5% blocking solution (5% FBS) 100ul, at 37 ℃ for two hours. Serum samples (1: 10, 1: 100, 1: 1000, 1: 10000, 1: 100000.) were diluted with PBS and 100. Mu.l/well were incubated in sealed ELISA plates for 1 hour at 37 ℃. PBST was washed 5 times with 260. Mu.l each, and 100. Mu.l of enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1: 5000 diluate) was added to each welld by PBS), incubated at 37 ℃ for 0.5 hour. The column was washed five times with PBST, 260. Mu.l each time, substrate TMB was added at 100. Mu.l/well, incubated for 15 minutes at room temperature in the dark, and the substrate was allowed to develop. Add 50. Mu.l of stop solution (2 NH) per well ₂ SO ₄ ) The color development was stopped and the plate was read with a microplate reader, OD450-630. And (3) a titer calculation method, namely selecting the maximum dilution factor with positive result, and multiplying the OD value/Cutoff value (0.1) corresponding to the dilution factor by the (X) dilution factor to obtain the value which is the titer of the antibody corresponding to the serum.

As a result: as shown in FIG. 29, compared with free EBV-gp350 protein, IFN alpha-Pan-EBV gp350-Fc protein can cause stronger specific humoral immune response in both single immunization (Prime) and double immunization (boost), which fully indicates that the vaccine platform can obviously and significantly compete for the immunogenicity of EBV-gp 350.

Example 19

IFNa-Pan-HSV-2 gD-Fc can cause stronger humoral immune response compared with free HSV-2 gD protein.

Materials: c57BL/6 female mice (6-8 weeks) were purchased from Beijing Wittingle Biotechnology, inc.; HSV-2 gD protein for ELISA was purchased from Baiying Biotech, inc. of Thai; HSV-2 gD protein for immunization was purchased from Baiying Biotechnology, inc. of Thai, and IFNa-Pan-HSV-2 gD-Fc was produced in the laboratory; other experimental materials were the same as in example three.

The method comprises the following steps:

(1) The mice are immunized by HSV-2 gD and IFNa-Pan-HSV-2 gD-Fc proteins, 10 mu g of IFNa-Pan-HSV-2 gD-Fc or HSV-2 gD proteins with the same molar amount are respectively mixed with 20 mu g of aluminum adjuvant overnight, then the mice are inoculated in a muscle immunization mode, and the boosting immunization is carried out for one time 14 days after the initial inoculation. Mouse sera were collected at day 14 and 28 after the primary immunization and assayed for levels of EBV-gp 350-specific antibodies by ELISA.

(2) Serum HSV-2 gD antibody was detected. Antigen coating: HSV-2 gD (1. Mu.g/ml) coating solution was added to an ELISA plate (Corning 9018) in a volume of 100. Mu.l per well and coated overnight at 4 ℃. Wash once with PBS, 260. Mu.l per well. Blocking was performed at 37 ℃ for two hours using 100. Mu.l of 5% blocking solution (5% FBS). Diluted with PBSSerum samples (1: 10, 1: 100, 1: 1000, 1: 10000, 1: 100000.) were released and 100. Mu.l/well were incubated in a closed ELISA plate for 1 hour at 37 ℃. PBST was washed 5 times with 260. Mu.l each, and 100. Mu.l of enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1: 5000diluted by PBS) was added to each well and incubated at 37 ℃ for 0.5 hour. The column was washed five times with PBST, 260. Mu.l each time, substrate TMB was added at 100. Mu.l/well, incubated for 15 minutes at room temperature in the dark, and the substrate was allowed to develop. Add 50. Mu.l of stop solution (2 NH) per well ₂ SO ₄ ) The color development was stopped and the plate was read with a microplate reader, OD450-630. And (3) a titer calculation method, namely selecting the maximum dilution factor with positive result, and multiplying the OD value/Cutoff value (0.1) corresponding to the dilution factor by the (X) dilution factor to obtain the value which is the titer of the antibody corresponding to the serum.

As a result: as shown in figure 30, IFNa-Pan-HSV-2 gD-Fc protein elicited stronger specific humoral immune responses in either single immunization (Prime) or double immunization (boost) compared to free HSV-2 gD protein, which is a good indication that this vaccine platform can significantly enhance the immunogenicity of HSV-2 gD.

Example 20: IFNa-Pan-VZV gE-Fc elicits a stronger humoral immune response than free VZV-2 gE protein and induces a Th1/Th2 balanced T cell immune response.

Materials: c57BL/6 female mice (6-8 weeks) were purchased from Beijing Wintolite Biotech, inc.; VZV gE protein for ELISA was purchased from baiying biotechnology limited, tezhou; VZV-gE protein for immunization was purchased from Baiying Biotech, inc. of Thai; detection of antibody subtype goat anti-mouse IgG1/IgG2c labeled with horseradish peroxidase (HRP) was purchased from proteintech; IFNa-Pan-VZV gE-Fc was produced in this laboratory; other experimental materials are the same as those in example three.

The method comprises the following steps:

(1) Mice were immunized with VZV gE, IFNa-Pan-VZV gE-Fc proteins, 10. Mu.g of IFNa-Pan-VZV gE-Fc or the same molar amount of VZV gE protein was mixed with 20. Mu.g of aluminum adjuvant overnight, and then the mice were immunized by intramuscular immunization and a booster immunization was performed 14 days after the initial immunization. Mouse sera were collected at 14 and 28 days after the primary immunization, and the VZV gE-specific IgG level in the mouse sera was measured by ELISA. Detection of VZV gE-specific antibody subtypes by ELISA 28 days after primary immunization

(2) Serum VSV gE total antibodies were detected. Antigen coating: VSV gE (1. Mu.g/ml) coating solution was added to ELISA plates (Corning 9018) in a volume of 100. Mu.l per well and coated overnight at 4 ℃. Wash once with PBS, 260. Mu.l per well. Blocking with 5% blocking solution (5% FBS) 100. Mu.l for two hours at 37 ℃. Serum samples (1: 10, 1: 100, 1: 1000, 1: 10000, 1: 100000.) were diluted with PBS and 100. Mu.l/well were incubated in sealed ELISA plates for 1 hour at 37 ℃. PBST was washed 5 times with 260. Mu.l each, 100. Mu.l of enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1: 5000diluted by PBS) was added to each well, and incubated at 37 ℃ for 0.5 hour. The column was washed five times with PBST, 260ul each time, substrate TMB 100. Mu.l/well was added, incubated at room temperature in the dark for 15 minutes, and the substrate was allowed to develop. Add 50. Mu.l of stop solution (2 NH) per well ₂ SO ₄ ) The color development was stopped and the plate was read with a microplate reader, OD450-630. And (3) selecting the maximum dilution multiple with positive result, and multiplying the OD value/Cutoff value (0.1) corresponding to the dilution multiple by the (X) dilution multiple to obtain the value which is the antibody titer corresponding to the serum.

(3) The serum VSV gE specific antibody subtype was detected. Antigen coating: VSV gE (1. Mu.g/ml) coating solution was added to ELISA plates (Corning 9018) in a volume of 100. Mu.l per well and coated overnight at 4 ℃. Wash once with PBS, 260. Mu.l per well. Blocking was performed at 37 ℃ for two hours using 100. Mu.l of 5% blocking solution (5% FBS). Serum samples (1: 10, 1: 100, 1: 1000, 1: 10000, 1: 100000) were diluted with PBS and 100. Mu.l per well were incubated in blocked ELISA plates for 1 hour at 37 ℃. PBST was washed 5 times, 260ul each time, 100. Mu.l of enzyme-labeled secondary antibody (horseradish peroxidase (HRP) -labeled goat anti-mouse IgG1/IgG2 c) was added to each well, and incubated at 37 ℃ for 0.5 hour. Washing with PBST five times, 260ul each time, adding substrate TMB 100 ul/well, incubating for 15 minutes at room temperature in dark place, and waiting for substrate color development. Add 50. Mu.l of stop solution (2 NH) per well ₂ SO ₄ ) The color development is stopped, and the plate is read by a microplate reader, OD450-630. The titer calculation method comprises selecting the maximum dilution factor with positive result, and multiplying the OD value/Cutoff value (0.1) corresponding to the dilution factor by the (X) dilution factor to obtain the value of the bloodCorresponding antibody titers were cleared.

As a result: compared with free VZV-gE protein, IFNa-Pan-VZV-gE-Fc protein can cause stronger specific humoral immune response (figure 31 a) in both single immunization (Prime) and double immunization (boost), IFNa-Pan-VZV-gE-Fc immunization can not only cause IgG1 with Th2 bias to generate, but also can cause IgG2c with high level Th1 bias to generate (figure 31 b-c), while VZV-gE immunization only induces IgG1 with Th2 bias to generate and cannot effectively generate IgG2c with Th1 bias, and the above results fully indicate that IFNa-Pan-VZV gE-Fc protein can not only generate stronger humoral immune response, but also can induce Th 1/2 balanced immune response compared with free VZV-gE protein.

Reference:

[1]WHO W.Global hepatitis report 2017[J].Geneva：World Health Organization，2017，

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

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

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

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

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

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

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

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

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

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

[12]MEKHAIEL D N，CZAJKOWSKY D M，ANDERSEN J T，et al.Polymetic human Fc-fusion proteins with modified effector functions[J].Scientific reports，2011，1(1)：1-11.

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

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

[15]BRACCI L，LA SORSA V，BELARDELLI F，et al.Type I interferons as vaccine adjuvants against infectious diseases and cancer[J].Expert review of vaccines，2008，7(3)：373-81.

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

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

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

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

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

[21]SPADARO F，LAPENTA C，DONATI S，et al.IFN-αenhances cross-presentation in human dendritic cells by modulating antigen survival，endocytic routing，and processing[J].Blood，The Journal of the American Society of Hematology，2012，119(6)：1407-17.

[22]PARLATO S，SANTINI S M，LAPENTA C，et al.Expression of CCR-7，MIP-3β，and Th-1 chemokines in type I IFN-induced monocyte-derived dendritic cells：importance for the rapid acquisition of potent migratory and functional activities[J].Blood，The Journal of the American Society of Hematology，2001，98(10)：3022-9.

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

[24]LE BON A，SCHIAVONI G，D′AGOSTINO G，et al.Type I interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo[J].Immunity，2001，14(4)：461-70.

Claims

1. a vaccine comprising a fusion protein having an interferon-target antigen-immunoglobulin Fc region (or antibody Ab) as a structural unit,

wherein the interferon is a first structural unit and may be a type I interferon, a type II interferon and/or a type III interferon such as IFN- α, IFN- β, IFN- γ, IFN-M (IL-29), IFN- λ 2 (IL-28 a), IFN- λ (IL-28 b) and IFN- ω, the interferons may be from human or murine origin, preferably the interferon is a type I interferon such as IFN- α, e.g. mouse IFN- α 4, human IFN- α 2, a mutant of human IFN- α 2 (which binds to human and mouse IFN receptors), e.g. as represented by the amino acid sequences of SEQ ID No.1, SEQ ID No.21, SEQ ID No.22,

wherein the target antigen is a third building block, which target antigen may be, for example, a tumor antigen, a pathogen antigen, such as a viral or bacterial antigen, wherein the target antigen may be, for example, a mutated target antigen different from the wild type, including, for example, a natural point mutation/deletion mutation/gain mutation/truncation of the wild type antigen, an artificial point mutation/deletion mutation/gain mutation/truncation, any combination of natural or artificial mutations, the resulting subtype after mutation, wherein the virus may be, for example, SARS-COV-2, or wherein the target antigen may be, for example, the full length or S1 region of the S protein of the SARS-COV-2 virus, for example, the target antigen may be an antigen as shown in SEQ ID No.76 or SEQ ID No.77,

wherein the immunoglobulin Fc region (or antibody) is a second structural unit, can be the constant region amino acid sequence of IgG1, igG2, igG3, igG4 and/or IgM, such as the Fc region of IgG1, and the Fc region shown by the amino acid sequences of IgG1-Fc-hole and IgG1-Fc-knob for heterodimer formation, SEQ ID NO.2, SEQ ID NO.23, and SEQ ID NO.24, wherein the antibody (including, for example, antibody heavy chain and light chain, or single chain antibody, ab for short) as a second structural unit can be DC targeted activation antibody, including anti-PD-L1, anti-DEC205, anti-CD80/86 and the like,

optionally the vaccine may be a targeted vaccine, optionally the fusion protein may further comprise one or more Th cell helper epitopes and/or linker fragments.

2. The vaccine of claim 1, wherein the target antigen is a viral antigen, which may be, for example, HBV, HPV, VZV, EBV, HSV-2, hiv, influenza virus, coronavirus, such as SARS-COV, SARS-COV-2, mers-CoY, e.g., the antigen may be an HBV antigen, such as HBV Pres1 antigen, HBsAg antigen or a peptide fragment, such as an ad subtype or ay subtype HBV Pres1 antigen, such as an ad subtype HBV Pres1 antigen as shown in the amino acid sequence of SEQ ID No.6, such as an ay HBV subtype Pres1 antigen as shown in the amino acid sequence of SEQ ID No. 26; for example, HBV HBsAg antigen (including various subtypes and peptide fragments), such as adr subtype HBV HBsAg antigen shown by an amino acid sequence of SEQ ID NO.7, adw subtype HBV HBsAg antigen shown by an amino acid sequence of SEQ ID NO.27, and ayw subtype HBV HBsAg antigen shown by an amino acid sequence of SEQ ID NO. 28; for example, the antigen can be, e.g., a SARS-COV-2 antigen, e.g., a SARS-COV 2RBD antigen as set forth in the amino acid sequence of SEQ ID NO. 8; for example, an influenza virus antigen, e.g., an influenza virus HA antigen, e.g., the influenza virus HA antigen shown in the amino acid sequence of SEQ ID No. 9; e.g., an HPV antigen, e.g., the HPV E7 antigen shown in the amino acid sequence of SEQ ID NO. 10; e.g., a gE antigen, such as the herpes zoster Virus (VZV) gE antigen shown in the amino acid sequence of SEQ ID No. 91; e.g., EBV-gp350, e.g., the Epstein-Barr virus (EBV) gp350 protein shown in the amino acid sequence of SEQ ID No. 92; for example, a gD antigen, such as the herpes simplex virus 2 (HSV-2) gD antigen represented by the amino acid sequence of SEQ ID NO. 93; the antigen may be, for example, EBV EBNA1/LMP2, VZV-IE62, HSV-2ICP0, HIV gp120 antigen.

Wherein the target antigen may be a mutated viral antigen, e.g. a mutant of any of the preceding viral antigens, e.g. a mutant of SARS-COV-2, including e.g. a natural point mutation/deletion mutation/gain mutation/truncation of a SARS-COV-2 protein (e.g. one or more of S protein, N protein, M protein, E protein), any combination of artificial point mutation/deletion mutation/gain mutation/truncation, any combination of natural or artificial mutations, a subtype resulting after mutation, e.g. the mutated viral antigen may be a mutant of the full length S protein (SEQ ID No. 76), the S1 region (SEQ ID No. 77), the RBD region (SEQ ID No. 78) of wild type SARS-COV-2, e.g. the mutated viral antigen may comprise one or more of the following mutations of the S protein of SARS-COV-2: NTD region 69-70 deletion, Y144 deletion, 242-244 deletion, L18F, D80A, D215, R246I mutation, RBD region K417, E484, N501Y, L452R mutation, D614G, H655Y mutation, for example the mutated viral antigen may comprise a mutation present in a mutant derived from British B.1.1.7 (501Y.1), south African B.1.351 (501Y.2) and Brazilian P1 (501Y.3), calif. B.1.429 mutant, for example the mutated viral antigen may comprise a mutant comprising a mutation shown in any of SEQ ID No.79, SEQ ID No.80, SEQ ID No.81, SEQ ID No.82, for example the mutated viral antigen may be a mutant comprising a sequence shown in any of SEQ ID No.79, SEQ ID No.80, SEQ ID No.81, SEQ ID No.82,

the viral antigen may be fusion expressed with a helper polypeptide epitope that enhances B cell and T cell responses, and may be located at the N-or C-terminus of the epitope, for example as represented by the Pan HLADR-binding epitope (PADER), for example as represented by the amino acid sequence of SEQ ID NO. 3;

the connecting segment of each structural unit is a flexible polypeptide sequence, can be connecting segments 1 and 2, and is shown as SEQ ID NO.4 and SEQ ID NO.25 amino acid sequences,

the N end of each polypeptide sequence consisting of the structural units can contain a corresponding signal peptide capable of promoting protein secretion, such as shown in an amino acid sequence of SEQ ID NO.5,

the vaccine may be produced by a eukaryotic expression system, for example by eukaryotic expression system 293F, CHO cells.

3. The vaccine of claim 1 or 2, wherein the target antigen is a tumor antigen, such as a tumor cell high expression protein molecule, for example, the antigen may be human epidermal growth factor receptor 2 (human epidermal growth factor receptor 2, HER2/neu) and Epidermal Growth Factor (EGFR); for example, tumor cells express high expression protein molecule Her2 and its functional regions and truncations, such as antigens shown in SEQ ID NO.85, 86, 97, 88, 89, 90 and mutants thereof.

4. The vaccine of any one of claims 1-3, wherein the fusion protein is a homodimer or heterodimer fusion protein, optionally the fusion protein may further comprise one or more Th cell helper epitopes and/or connecting fragments in either or both strands (i.e. the first and/or second polypeptide chain) of the homodimer or heterodimer,

optionally the homodimer fusion protein comprises a first polypeptide chain and a second polypeptide chain that are identical, e.g., the first and second polypeptide chains comprise from N-terminus to C-terminus an IFN, a target antigen and an immunoglobulin Fc region (or Ab), or a polypeptide of any combined order of the three structural units, and generate homodimers; preferably comprising IFN, a target antigen and an immunoglobulin Fc region (or Ab) in sequence from N-terminus to C-terminus; it may also comprise a fusion protein of Th cell helper epitopes;

optionally said heterodimeric fusion protein comprises a first polypeptide chain and a second polypeptide chain, said first and second polypeptide chains being different, e.g., said first polypeptide chain can comprise from N-terminus to C-terminus an IFN and an immunoglobulin Fc region (or Ab), or from N-terminus to C-terminus an immunoglobulin Fc region (or Ab) and an IFN, said second polypeptide chain can comprise a target antigen and an immunoglobulin Fc region (or Ab), wherein the target antigen can be at the N-terminus, the immunoglobulin region (or Ab) can be at the C-terminus, or the immunoglobulin region (or Ab) can be at the N-terminus, the target antigen can be at the C-terminus; or three structural units in any combination order, and generating heterodimers; preferably, the IFN and target antigenic sites are located at the N-terminus of the two polypeptides, respectively, and the immunoglobulin Fc region (or Ab) is located at the C-terminus of the two polypeptides; it may also comprise a fusion protein of Th cell helper epitopes.

5. The vaccine of claim 4, wherein

1) The first and second polypeptides of the homodimer may comprise the amino acid sequences shown in SEQ ID No.11, 12, 13, 14, 29, 30, 31, 32, 38, 39, 40, 47, 48, 49, 50, 51, 56, 57, 59, 58, 65, 66, 67, 68,

2) The first heterodimeric polypeptide may comprise the nucleotide sequence shown in SEQ ID No.15, 33, 42, 51, 60, 69, and the second polypeptide comprises the amino acid sequence shown in SEQ ID No.16, 17, 18, 19, 34, 35, 36, 37, 43, 44, 45, 46, 52, 53, 54, 55, 61, 62, 63, 64, 70, 71, 72, 73,

3) The antibody may include DC targeting antibody, immune checkpoint blocking antibody, immune activating antibody, etc., such as vaccine comprising antibody amino acid sequence of anti-PD-L1 antibody (SEQ ID NO. 20), anti-DEC205 antibody, anti-CD80/86, etc.

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

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

8. The use according to claim 7, wherein the composition or kit is for the prevention or treatment of tumors or pathogens, e.g. the prevention or treatment of viruses or bacteria, which may be HBV, HPV, EBV, influenza, HIV, coronaviruses, such as SARS-COV, SARS-COV-2, MERS-CoV, e.g. the composition or kit is for use as a hepatitis B prophylactic or therapeutic vaccine, HBV prophylactic or therapeutic vaccine, influenza prophylactic or therapeutic vaccine, SARS-COV2 prophylactic or therapeutic vaccine, HPV-related tumor prophylactic or therapeutic vaccine, EBV-related tumor prophylactic or therapeutic vaccine, HIV prophylactic or therapeutic vaccine.

9. The vaccine of any one of claims 1 to 5 or the use of claim 7 or 8, wherein the vaccine, the composition or the kit is administered intramuscularly, intravenously, transdermally, subcutaneously or intranasally, wherein the vaccine, the composition or the kit further comprises an adjuvant which may comprise aluminium adjuvant (Alum), toll-like receptor 4 activator ligand MPLA, toll-like receptor 9 ligand, oligodeoxynucleotide (CpG-ODN), MF59 and Freund's adjuvant.

10. The vaccine according to any one of claims 1-5 or the use according to claim 7 or 8, wherein the vaccine may be used in combination with a further prophylactic or therapeutic treatment, e.g. the vaccine may be an HBV therapeutic vaccine which may be used in combination with a further prophylactic or therapeutic HBV treatment, e.g. the HBV therapeutic vaccine may be used in combination with a hepatitis b virus envelope protein HBsAg vaccine, e.g. for the treatment of chronic hepatitis b virus infection, e.g. the HBV therapeutic vaccine may be used in combination with a nucleoside or nucleotide analogue, e.g. for the treatment of chronic hepatitis b virus infection, e.g. for the treatment of influenza, SARS-COV2, HPV, EBV, HIV prophylactic or therapeutic vaccine, etc. in combination with an antiviral drug and a further therapeutic approach; use of a vaccine for prevention or treatment of HPV, EBV-related tumours in combination with an anti-viral anti-tumour drug and therapy, e.g. any vaccine according to any one of claims 1 to 5 as a component of a vaccine in combination with a multivalent combination vaccine comprising another virus or pathogen or tumour vaccine, e.g. any multivalent vaccine comprising a SARS-COV-2 vaccine according to any one of claims 1 to 5 in combination with an influenza vaccine or other vaccine, e.g. any vaccine according to any one of claims 1 to 5 in combination with an adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of the same virus, pathogen, tumour, e.g. a SARS-COV-2 fusion protein vaccine in combination with an adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of SARS-COV-2 in a sequential or simultaneous immunization schedule, e.g. the sequential or simultaneous immunization schedule may be: 1) Firstly, the SARS-COV-2 fusion protein vaccine is immunized and then immunized; 2) Firstly, immunizing an adenovirus vaccine or an mRNA vaccine or an inactivated vaccine or a DNA vaccine of SARS-COV-2, and then immunizing an ARS-COV-2 fusion protein vaccine; 3) The SARS-COV-2 fusion protein vaccine and SARS-COV-2 adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine are simultaneously immunized.