CN118320111A - Nanoparticle vaccine of EB virus and preparation and application of nanoparticle vaccine and combination thereof - Google Patents

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the preparation and application of a nanoparticle vaccine of Epstein-Barr virus and its combination. The present invention provides four immunogenic complexes, which improve the immunogenicity of the corresponding immunogens by granulating the immunogens, and significantly stimulate humoral and cellular immune responses. The mouse serum after immunizing mice with the nanoparticle vaccine prepared by the present invention can effectively neutralize EBV-infected B cells in vitro. Compared with the ungranulated vaccine, the total anti-titer is increased by 14 to 43 times, and the neutralization titer for B cells is increased by 19 to 58 times. The nanoparticle vaccine can effectively stimulate the generation of immune memory. The present invention also provides a nanoparticle combination vaccine, and the neutralization titer for B cells is about 11 times, 4 times, 9 times and 16 times higher than that of the single gp350ECD ₁₂₃ , gp42, gHgL, and gB nanoparticle vaccines, respectively.

Description

Preparation and application of a nanoparticle vaccine for Epstein-Barr virus and its combination

Technical Field

The present invention belongs to the field of biomedicine technology, and specifically relates to the preparation and application of a nanoparticle vaccine of Epstein-Barr virus and a combination thereof.

Background technique

Epstein-Barr virus (EBV) is widely infected in the global population. It is reported that about 90% of adults have been infected with EBV. Infection with EBV in adolescence is likely to cause mononucleosis, which can generally heal on its own; people who have been infected with EBV are generally lifelong carriers of the virus. EBV is the first virus that has been proven to induce tumors after infection. More and more evidence shows that EBV is closely related to the occurrence and development of various lymphoid tumors (including Hodgkin's lymphoma, Burkitt's lymphoma, NK/T cell lymphoma, etc.) and epithelial tumors (including nasopharyngeal carcinoma, gastric cancer, etc.). In addition, EBV infection is closely related to autoimmune diseases such as multiple sclerosis. However, to date, there is no specific treatment for diseases such as cancer induced by EBV, and there is no effective preventive vaccine against EBV infection available on the market. Therefore, it is particularly important to develop a vaccine to prevent EBV infection.

gp350 is the most abundant glycoprotein on the surface of EBV and plays an important role in the process of EBV infection of B cells. Antibodies targeting gp350 are the most important antibodies that neutralize EBV-infected B cells in healthy EBV carriers. The receptor of gp350 is CR2, and the binding site is located at the N-terminal ECD123.

gB is a membrane fusion glycoprotein expressed by EBV. It belongs to the type III membrane fusion protein and exists on the surface of the viral envelope in the form of a homotrimer. After EBV recognizes and binds to multiple cell receptors, the cascade triggers the membrane fusion function of gB, and ultimately completes the fusion of the viral envelope and the cell membrane through conformational changes, successfully realizing the virus's infection and entry into the cell.

The gHgL protein is a heterodimer located in the EBV envelope, in which gH is a single transmembrane protein and gL is a secretory protein. gHgL is involved in the process of EBV infection of B cells and epithelial cells and binds to the epithelial cell receptor integrin or EphA2.

gp42 is also a single-pass transmembrane protein with its N-terminus located inside the cell and its C-terminus located outside the cell. It binds to the receptor HLA-II and participates in the process of EBV infection of B cells. In this process, it forms a heterotrimer with the gHgL protein to exert its effect.

EBV gp350, gB, gHgL and gp42 proteins are the main glycoproteins involved in EBV infection of B cells or epithelial cells, and are also the most studied EBV membrane surface glycoproteins. During natural infection, they can stimulate the body to produce specific immune responses against EBV at the serum and cellular immune levels. However, the immunogenicity of subunit vaccines is low and needs to be further improved.

Summary of the invention

The first aspect of the present invention aims to provide an immunogenic complex.

The second aspect of the present invention aims to provide a biomaterial.

The third aspect of the present invention is to provide a method for preparing an immunogenic complex.

The fourth aspect of the present invention aims to provide an application.

The fifth aspect of the present invention aims to provide a medicine.

The sixth aspect of the present invention aims to provide a vaccine.

The seventh aspect of the present invention aims to provide a complete kit.

In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

The first aspect of the present invention provides an immunogenic complex comprising:

A recombinant protein A containing an antigen; and a recombinant protein B containing a carrier protein;

The antigen is selected from any one of gHgL, gB, gp350, and gp42 of Epstein-Barr virus;

The carrier protein is Ferritin;

The recombinant protein A and the recombinant protein B are covalently linked via SpyCatcher-SpyTag.

Preferably, the immunogenic complex is any one of immunogenic complex A, immunogenic complex B, immunogenic complex C, and immunogenic complex D:

Preferably, the immune complex A comprises: a recombinant protein A containing an antigen; and a recombinant protein B containing a carrier protein; and the antigen is gHgL of Epstein-Barr virus.

Preferably, the immune complex B comprises: a recombinant protein A containing an antigen; and a recombinant protein B containing a carrier protein; and the antigen is gB of Epstein-Barr virus.

Preferably, the immune complex C comprises: a recombinant protein A containing an antigen; and a recombinant protein B containing a carrier protein; and the antigen is gp350 of Epstein-Barr virus.

Preferably, the immune complex D comprises: a recombinant protein A containing an antigen; and a recombinant protein B containing a carrier protein; and the antigen is gp42 of Epstein-Barr virus.

Preferably, the recombinant protein A comprises an antigen and a SpyCatcher, and the recombinant protein B comprises a carrier protein and a SpyTag.

Preferably, the recombinant protein A comprises an antigen and a SpyTag, and the recombinant protein B comprises a carrier protein and a SpyCatcher.

Preferably, the antigen is connected to the SpyCatcher or SpyTag via a connecting peptide; or the carrier protein is connected to the SpyCatcher or SpyTag via a connecting peptide.

Preferably, the number of amino acids in the connecting peptide is 1 to 30; it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

Preferably, the connecting peptide is a meaningless polypeptide having no additional functions other than connection (eg, protein localization, enzyme cleavage site, etc.).

Preferably, the connecting peptide is a flexible connecting peptide.

Preferably, the amino acids in the connecting peptide are selected from one or more of Gly, Ser, Pro, Ala and Glu.

Preferably, the connecting peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n, (GSG)n or (G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6;

Wherein (GGS)n indicates that there are n GGS repeats, for example, (GGS)4 indicates GGSGGSGGSGGS (SEQ ID NO.1), (GSG)3 indicates GSGGSGGSG (SEQ ID NO.2), and the same applies to the others.

Preferably, the amino acid sequence of the flexible connecting peptide is (GGGGS)3, as shown in SEQ ID NO.3;

Preferably, the recombinant protein A comprises the SpyCatcher, the connecting peptide and the antigen in sequence from the N-terminus to the C-terminus.

Preferably, the recombinant protein B comprises the SpyTag, the connecting peptide and the carrier protein in sequence from the N-terminus to the C-terminus.

Preferably, the recombinant protein A further comprises a tag sequence.

Preferably, the recombinant protein B further comprises a tag sequence.

Preferably, the tag sequence includes at least one of His, Myc, HA, Flag, and GST.

Preferably, the tag sequence is located at the N-terminus and/or C-terminus of recombinant protein A.

Preferably, the tag sequence is located at the C-terminus of recombinant protein A.

Preferably, the tag sequence is located at the N-terminus and/or C-terminus of recombinant protein B.

Preferably, the tag sequence is located at the C-terminus of recombinant protein B.

Preferably, the gHgL comprises gL and gH; further comprises a gL protein extramembrane region sequence of 24 to 137AA and a gH protein extramembrane region sequence of 19 to 678AA, as shown in SEQ ID NO.4.

Preferably, the gB includes the amino acid sequence 24 to 683AA of the gB protein extracellular domain; further, the amino acids at positions 112-113 of the gB are replaced from WY to HR, and the amino acids at positions 193-196 are replaced from WLIW to RVEA, as shown in SEQ ID NO.5.

Preferably, the gp350 is gp350 ECD ₁₂₃ ; further, it is region 2 to 425AA of ECD123 of the gp350 protein, as shown in SEQ ID NO.6.

Preferably, the gp42 is the gp42 protein extracellular domain sequence 34 to 223AA, as shown in SEQ ID NO.7.

Preferably, the amino acid sequence of the carrier protein Ferritin is shown in SEQ ID NO.8.

Preferably, the amino acid sequence of the SpyCatcher is shown in SEQ ID NO.9.

Preferably, the amino acid sequence of the SpyTag is as shown in SEQ ID NO.10.

Preferably, when the antigen is gHgL, the amino acid sequence of the recombinant protein A is as shown in SEQ ID NO.11;

Preferably, when the antigen is gB, the amino acid sequence of the recombinant protein A is as shown in SEQ ID NO.12:

Preferably, when the antigen is gp350, the amino acid sequence of the recombinant protein A is as shown in SEQ ID NO.13:

Preferably, when the antigen is gp42, the amino acid sequence of the recombinant protein A is as shown in SEQ ID NO.14:

Preferably, the amino acid sequence of the recombinant protein B is as shown in SEQ ID NO.15.

The second aspect of the present invention provides a biological material related to the immunogenic complex of the first aspect of the present invention, wherein the biological material is any one of B1) to B12):

B1) a nucleic acid molecule encoding the immunogenic complex of any one of claims 1 to 3;

B2) an expression cassette containing the nucleic acid molecule described in B1);

B3) a recombinant vector containing the nucleic acid molecule described in B1);

B4) a recombinant vector containing the expression cassette described in B2);

B5) a recombinant microorganism containing the nucleic acid molecule described in B1);

B6) a recombinant microorganism containing the expression cassette described in B2);

B7) a recombinant microorganism containing the recombinant vector described in B3);

B8) a recombinant microorganism containing the recombinant vector described in B4);

B9) a transgenic animal cell line containing the nucleic acid molecule described in B1);

B10) a transgenic animal cell line containing the expression cassette described in B2);

B11) a transgenic animal cell line containing the recombinant vector described in B3);

B12) A transgenic animal cell line containing the recombinant vector described in B4).

The third aspect of the present invention provides a method for preparing the immunogenic complex of the first aspect of the present invention, comprising incubating recombinant protein A with recombinant protein B to obtain.

Preferably, the molar mass ratio of the recombinant protein A to the recombinant protein B is (6-10):1.

Preferably, the incubation time is 4-24 hours.

Preferably, the incubation time is 10-14 hours.

Preferably, the incubation temperature is 1-25°C.

Preferably, the incubation temperature is 1-8°C.

The fourth aspect of the present invention provides use of the immunogenic complex of the first aspect of the present invention and/or the biomaterial of the second aspect of the present invention in the preparation of a drug.

Preferably, the drug has at least one of the functions c1)-c2):

c1) Prevention of EB virus infection;

c2) Treating and/or preventing diseases caused by EB virus infection.

Preferably, the disease caused by Epstein-Barr virus infection includes infectious mononucleosis.

The fifth aspect of the present invention provides a drug comprising the immunogenic complex of the first aspect of the present invention and/or the biological material of the second aspect of the present invention.

Preferably, the drug further comprises pharmaceutically acceptable excipients.

Preferably, the auxiliary material comprises at least one of an excipient, a preservative, an antibacterial agent, a flavoring agent, a stabilizer, a disintegrant, and a lubricant.

The sixth aspect of the present invention provides any one of the vaccines a1) to a2):

a1) a vaccine comprising the immunogenic complex of the first aspect of the invention and/or the biological material of the second aspect of the invention;

a2) A vaccine comprising at least two of the immunogenic complex A, immunogenic complex B, immunogenic complex C, and immunogenic complex D according to the first aspect of the present invention.

Preferably, the vaccine described in a2) comprises the immunogenic complex A, immunogenic complex B, immunogenic complex C, and immunogenic complex D of the first aspect of the present invention.

Preferably, the vaccine described in a1) and a2) further comprises: an adjuvant.

The seventh aspect of the present invention provides a kit comprising the vaccine of the sixth aspect of the present invention and a container for vaccination.

The beneficial effects of the present invention are:

The present invention uses ferritin as a carrier protein, and uses spytag-spycatcher technology to display gHgL, gB, gp350ECD123 and gp42 proteins on the surface of Ferritin, respectively, to obtain four immunogenic complexes, while ensuring the correct modification and folding of the antigen and achieving high expression. After the Ferritin nanoparticle vaccine was immunized with mice, the humoral immune response was significantly stimulated, and the total anti-titer was increased by 14 to 43 times compared with the non-granulated vaccine. The immunized mouse serum can effectively neutralize EBV-infected B cells in vitro, and the neutralization titer against B cells is increased by 19 to 58 times compared with the non-granulated vaccine. Secondly, the nanoparticle vaccine can significantly stimulate the generation of antigen-specific cellular immune responses, which is the first time to illustrate the superiority of nanoparticle vaccines in stimulating cellular immune responses. In addition, the present invention reports for the first time that the Ferritin nanoparticle EBV vaccine has a stronger ability to stimulate immune memory. Compared with the non-granulated vaccine, the nanoparticle vaccine immunization significantly stimulates the generation of memory T cells and memory B cells.

In addition, the nanoparticle vaccine prepared by the present invention can effectively stimulate the generation of immune memory. When the nanoparticle vaccine is used in combination, the neutralization titer against B cells is about 11 times, 4 times, 9 times and 16 times higher than that of the single gp350 immunogenic complex, gp42 immunogenic complex, gHgL immunogenic complex and gB immunogenic complex, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the purification and assembly of the components of the immunogenic complex: A is a schematic diagram of the structure of the components of the immunogenic complex; B is the protein purification and assembly results, st represents SpyTag, sc represents SpyCatcher, F represents Ferritin, and gp350 represents gp350ECD ₁₂₃ ; C is a schematic diagram of the 3D structure simulation of the components of the immunogenic complex.

FIG2 shows the morphology of the nanoparticle vaccine under transmission electron microscopy: F represents Ferritin, and gp350 represents gp350ECD ₁₂₃ .

FIG3 shows the particle size of the nanoparticle vaccine: F represents Ferritin, and gp350 represents gp350ECD ₁₂₃ .

Figure 4 shows the reactivity of nanoparticles or monomers of various proteins with specific antibodies: A represents the binding of gHgL nanoparticles or monomers with different antibodies; B represents the binding of gp350ECD ₁₂₃ nanoparticles or monomers with different antibodies; C represents the binding of gp42 nanoparticles or monomers with different antibodies; and D represents the binding of gB nanoparticles or monomers with different antibodies.

Figure 5 shows the humoral immune response of mice activated by nanoparticle vaccines and their control vaccines: A represents the total antibody binding titer test results of different nanoparticle vaccines in mouse serum; B represents the results of the serum neutralizing B cell infection ability after immunization with different nanoparticle vaccines.

Figure 6 shows the cellular immune response of mice activated by nanoparticle vaccines and their control vaccines: A represents the activation effect of different nanoparticle vaccines on TNF-α of mouse CD4 ⁺ T cells; B represents the activation effect of different nanoparticle vaccines on TNF-α of mouse CD8 ⁺ T cells.

Figure 7 shows the immune memory mediated by nanoparticle vaccines and their control vaccines: A represents the immune memory results of different nanoparticle vaccines on mouse CD4 effector T cells; B represents the immune memory results of different nanoparticle vaccines on mouse CD8 effector T cells; C represents the effect of different nanoparticle vaccines on the number of mouse memory B cells.

Figure 8 Neutralization titer after combined immunization with nanoparticle vaccines.

Detailed ways

The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

As used herein, the term "particle" refers to a geometric body having a specific shape within a size range, a state of matter characterized by the presence of discrete particles, pellets, beads or agglomerates, regardless of their size, shape or morphology.

As used herein, the term "nanoparticle" refers to a particle having one dimension of size (ie, the diameter in the longest dimension of the particle) that is less than 100 nanometers.

As used in this article, the term "particle size" or "equivalent particle size" means that when a certain physical property or physical behavior of the measured particle is closest to a homogeneous sphere (or combination) of a certain diameter, the diameter (or combination) of the sphere is taken as the equivalent particle size (or particle size distribution) of the measured particle.

As used herein, the term "average particle size" refers to a real particle group consisting of particles of different sizes and shapes, compared with a hypothetical particle group consisting of uniform spherical particles, if the particle diameters of the two are the same, then the diameter of the spherical particles is called the average particle size of the real particle group. The method for measuring the average particle size is known to those skilled in the art, such as the light scattering method; the instrument for measuring the average particle size includes but is not limited to the Malvern particle sizer.

As used herein, the term "room temperature" refers to 25±5°C.

As used herein, the term "immune adjuvant" refers to a substance that is administered to the body together with an antigen or in advance to enhance immunogenicity or change the type of immune response. The immune adjuvant itself can be immunogenic (e.g., BCG), or non-immunogenic (e.g., aluminum hydroxide adjuvant).

As used herein, the term "antigen" or "immunogen" refers to a substance that is capable of inducing a specific immune response in a host. Antigens may include a whole organism (e.g., an inactivated, attenuated, or live organism); a subunit or portion of an organism; a recombinant vector containing an immunogenic insert; a portion or fragment of DNA that is capable of inducing an immune response after presentation to a host; a protein, glycoprotein, lipoprotein, polypeptide, peptide, antigenic epitope, hapten, toxin, antitoxin, or any combination thereof. Example 1 Preparation of SpyTag-Ferritin Protein

The N-terminus of the Ferritin protein (amino acid sequence as shown in SEQ ID NO.8) was connected to the SpyTag protein (amino acid sequence as shown in SEQ ID NO:10) through a flexible amino acid sequence (SEQ ID NO.3), and the C-terminus of Ferritin was connected to a polyhistidine polypeptide (6×His; SEQ ID NO.16) for affinity chromatography purification. The above sequence was constructed into the prokaryotic expression vector PET-28a, and the successfully constructed recombinant plasmid was transformed into BL21. After induction with 0.5mM IPTG at 25°C for 12h, the supernatant was collected after ultrasonic disruption, and the supernatant was obtained after heat precipitation at 65°C for 30min, and then the supernatant was mixed with Ni The protein was incubated overnight with 6Fast Flow (GE) medium and purified after elution with 500 mM imidazole (25 mM Hepes, 250 mM NaCl) to obtain the SpyTag-Ferritin protein, whose amino acid sequence is shown in SEQ ID NO.15.

Example 2 Preparation of Ferritin-gHgL Immunogenic Complex

With reference to the complete gene sequence of EB virus M81 strain (KF373730.1), the C-terminus of the gL protein extramembrane region sequence (24-137AA, the amino acid sequence is shown in SEQ ID NO.17) was connected to the gH protein extramembrane region sequence (19-678AA, the amino acid sequence is shown in SEQ ID NO.18) through a flexible amino acid sequence (SEQ ID NO.3), the N-terminus of the gL protein was connected to the spycatcher protein (the amino acid sequence is shown in SEQ ID NO.9) through a flexible amino acid sequence (SEQ ID NO.3), the N-terminus of the spycatcher was connected to the signal peptide coding sequence MPMGSLQPLATLYLLGMLVASCLG (N-terminus to C-terminus, SEQ ID NO.19), and the C-terminus of the gH protein was connected to a tag protein (6×His, SEQ ID NO.9) for easy affinity chromatography purification. The above sequences were constructed into a eukaryotic expression vector pCDNA3.1(+), and the successfully constructed recombinant plasmid was transfected into 293F cells. After 5 days of expression, the supernatant was collected and mixed with Ni 6Fast Flow (GE) medium was incubated overnight, and spycatcher-gHgL protein was finally obtained after elution with 500mM imidazole (25mM Hepes, 250mM NaCl). The amino acid sequence of spycatcher-gHgL protein is shown in SEQ ID NO.11: N-VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRD SSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGGGSGGGGSGGGGSSWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCANGLNVVSFFISILKRSSSALTSHLRELLTTLESLYGSFSVEDLFGANLNRYAWHRGGGGGGSGGGGSGGGGSSSLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEAL-C.

The SpyTag-Ferritin protein prepared in Example 1 was incubated with the spycatcher-gHgL protein, the incubation conditions were 4°C, 12h, the incubation buffer was PBS, and the molar mass ratio of the spycatcher-gHgL protein to the SpyTag-Ferritin protein was 8: 1. After incubation, the excess spycatcher-gHgL protein was purified by molecular sieve (Superdex 200Increase 10/300GL columns) to obtain the Ferritin-gHgL immunogenic complex.

Example 3 Preparation of Ferritin-gB Immunogenic Complex

The WY112–113 and WLIW193–196 in the amino acid sequence of the gB protein of the EBV M81 strain (KF373730.1) were replaced with HR177–178 and RVEA258–261 in the amino acid sequence of the HSV-1 gB protein, respectively. The amino acid sequence of the gB protein extracellular domain after replacement (24-683AA) is shown in SEQ ID NO.5. The N-terminus was connected to the spycatcher protein (amino acid sequence as shown in SEQ ID NO.9) through a flexible amino acid sequence (SEQ ID NO.3), the spycatcher N-terminus was connected to the signal peptide coding sequence MPMGSLQPLATLYLLGMLVASCLG (N-terminus to C-terminus, SEQ ID NO.19), and the gB protein C-terminus was connected to a polyhistidine polypeptide (6×His, SEQ ID NO.16) that was convenient for affinity chromatography purification. The above sequences were constructed into the eukaryotic expression vector pCDNA3.1(+), and the successfully constructed recombinant plasmid was transfected into 293F cells. After 5 days of expression, the supernatant was collected and mixed with Ni After overnight incubation with 6Fast Flow (GE) medium, the spycatcher-gB protein was finally obtained after elution with 500mM imidazole (25mM Hepes, 250mM NaCl). The amino acid sequence of the spycatcher-gB protein is shown in SEQ ID NO.12: N-VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISD-C.

The SpyTag-Ferritin protein prepared in Example 1 was incubated with the spycatcher-gB protein, the incubation conditions were 4°C, 12h, the incubation buffer was PBS, and the molar mass ratio of the spycatcher-gB protein to the SpyTag-Ferritin protein was 8: 1. After incubation, the excess spycatcher-gB protein was purified by molecular sieve (Superdex 200Increase 10/300GL columns) to obtain the Ferritin-gB immunogenic complex.

Example 4 Preparation of Ferritin-gp350ECD ₁₂₃ Immunogenic Complex

The ECD123 region (2-425AA; SEQ ID NO.6) of the gp350 protein of the EBV M81 strain (KF373730.1) was connected to the spycatcher protein (SEQ ID NO.9) at the N-terminus through a flexible amino acid sequence (SEQ ID NO.3), the spycatcher N-terminus was connected to the signal peptide coding sequence MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGAR (N-terminus to C-terminus, SEQ ID NO.20), and the gp350 protein C-terminus was connected to a polyhistidine polypeptide (6×His, SEQ ID NO.16) that was convenient for affinity chromatography purification. The above sequences were constructed into a suitable eukaryotic expression vector pVRC8400, and the successfully constructed recombinant plasmid was transfected into 293F cells. After 5 days of expression, the supernatant was collected and mixed with Ni After overnight incubation with 6Fast Flow (GE) medium, the spycatcher-gp350ECD ₁₂₃ protein was finally obtained after elution with 500 mM imidazole (25 mM Hepes, 250 mM NaCl), and its amino acid sequence is shown in SEQ ID NO.13: N-VDTLSGLSSEQGQSGDMTIEED-C.

The SpyTag-Ferritin protein prepared in Example 1 was incubated with the spycatcher-gp350ECD ₁₂₃ protein, the incubation conditions were 4°C, 12h, the incubation buffer was PBS, and the molar mass ratio of the spycatcher-gp350ECD ₁₂₃ protein to the SpyTag-Ferritin protein was 8: 1. After incubation, the excess spycatcher-gp350ECD ₁₂₃ protein was purified by molecular sieve (Superdex 200Increase 10/300GL columns) to obtain the Ferritin-gp350ECD ₁₂₃ immunogenic complex.

Example 5 Preparation of Ferritin-gp42 Immunogenic Complex

The N-terminus of the extracellular domain sequence (34-223AA; SEQ ID NO.4) of the gp42 protein of the EBV M81 strain (KF373730.1) was connected to the spycatcher protein (SEQ ID NO.9) through a flexible amino acid sequence (SEQ ID NO.3), the N-terminus of the spycatcher was connected to the signal peptide coding sequence MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGAR (N-terminus to C-terminus, SEQ ID NO.20), and the C-terminus of the gp42 protein was connected to a polyhistidine polypeptide (6×His, SEQ ID NO.16) that was convenient for affinity chromatography purification. The above sequences were constructed into a suitable eukaryotic expression vector pVRC8400, and the successfully constructed recombinant plasmid was transfected into 293F cells. After 5 days of expression, the supernatant was collected and mixed with Ni 6Fast Flow (GE) medium was incubated overnight, and spycatcher-gp42 protein was finally obtained after elution with 500mM imidazole (25mM Hepes, 250mM NaCl). The amino acid sequence of the protein is shown in SEQ ID NO.14: N-VDTLSGLSSEQGQSGDMTIEEDSATHIKFSK RDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGGGSGGGGSGGGGSGGRVAAAAITWVPKPNVEVWPVDPPPPVNFNKTAEQEYGDKEVKLPHWTPTLHTFQVPQNYTKANCTYCNTREYTFSYKGCCFYFTKKKHTWNGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTFKVYQIFGSHCTYVSKFSTVPVSHHECSFLKPCLCVSQRSNSHHHHHH-C.

The SpyTag-Ferritin protein prepared in Example 1 was incubated with the spycatcher-gp42 protein, the incubation conditions were 4°C, 12h, the incubation buffer was PBS, and the molar mass ratio of the spycatcher-gp42 protein to the SpyTag-Ferritin protein was 8: 1. After incubation, the excess spycatcher-gp42 protein was purified by molecular sieve (Superdex 200Increase 10/300GL columns) to obtain the Ferritin-gp42 immunogenic complex.

Example 6 Preparation of Ferritin-gHgL Nanoparticle Vaccine

5 μg of the Ferritin-gHgL immunogenic complex prepared in Example 2 was mixed with CpG adjuvant to obtain 50 μL of CpG adjuvant-combined Ferritin-gHgL, which was further mixed with 50 μL of aluminum adjuvant to obtain 100 μL of Ferritin-gHgL nanoparticle vaccine (Alum+CpG adjuvant-combined Ferritin-gHgL, A-F-gHgL).

The usage of CpG adjuvant (Invivogen, Cat no: vac-2395-1) and aluminum adjuvant (lmject Alum Adjuvant, Thermo, Cat no: 77161) was described in the instructions.

Example 7 Preparation of Ferritin-gB Nanoparticle Vaccine

5 μg of the Ferritin-gB immunogenic complex prepared in Example 3 was mixed with CpG adjuvant to obtain 50 μL of CpG adjuvant-combined Ferritin-gB, which was further mixed with 50 μL of aluminum adjuvant to obtain 100 μL of Ferritin-gB nanoparticle vaccine (Alum+CpG adjuvant-combined Ferritin-gB, A-F-gB).

The usage of CpG adjuvant (Invivogen, Cat no: vac-2395-1) and aluminum adjuvant (lmject Alum Adjuvant, Thermo, Cat no: 77161) was described in the instructions.

Example 8 Preparation of Ferritin-gp350ECD ₁₂₃ Nanoparticle Vaccine

5 μg of the Ferritin-gp350ECD ₁₂₃ immunogenic complex prepared in Example 4 was mixed with CpG adjuvant to obtain 50 μL of CpG adjuvant-combined Ferritin-gp350ECD ₁₂₃ , which was further mixed with 50 μL of aluminum adjuvant to obtain 100 μL of Ferri-tin-gB nanoparticle vaccine (Alum+CpG adjuvant-combined Ferritin-gp350ECD ₁₂₃ , AF-gp350ECD ₁₂₃ ).

The usage of CpG adjuvant (Invivogen, Cat no: vac-2395-1) and aluminum adjuvant (lmject Alum Adjuvant, Thermo, Cat no: 77161) was described in the instructions.

Example 9 Preparation of Ferritin-gp42 Nanoparticle Vaccine

5 μg of the Ferritin-gp42 immunogenic complex prepared in Example 4 was mixed with CpG adjuvant to obtain 50 μL of CpG adjuvant-combined Ferritin-gp42, which was further mixed with 50 μL of aluminum adjuvant to obtain 100 μL of Ferritin-gp42 nanoparticle vaccine (Alum+CpG adjuvant-combined Ferritin-gp42, A-F-gp42).

The usage of CpG adjuvant (Invivogen, Cat no: vac-2395-1) and aluminum adjuvant (lmject Alum Adjuvant, Thermo, Cat no: 77161) was described in the instructions.

Example 10 Preparation of a Nanoparticle Combined Vaccine

A total of 5 μg (mass ratio of 1:1:1:1) of Ferritin-gHgL, Ferritin-gB, Ferritin-gp350ECD ₁₂₃ , and Ferritin-gp42 prepared in Example 2-5 were mixed with CpG adjuvant to obtain 50 μL of CpG adjuvant. Then, 50 μL of aluminum adjuvant was added to obtain 100 μL of nanoparticle combination vaccine (Alum+CpG adjuvant Ferritin-gHgL, Ferritin-gB, Ferritin-gp350ECD ₁₂₃ , Ferritin-gp42, AF-cocktail).

The usage of CpG adjuvant (Invivogen, Cat no: vac-2395-1) and aluminum adjuvant (lmject Alum Adjuvant, Thermo, Cat no: 77161) was described in the instructions.

Comparative Example 1 Preparation of soluble HgL vaccine

The preparation method of gHgL protein is the same as that of Example 2, except that gHgL is not linked to Ferritin protein via SpyCatcher-Spytag, and its amino acid sequence is shown in SEQ.ID NO.21.

The preparation method of gHgL soluble vaccine was the same as that in Example 6, and 100 μL of gHgL soluble vaccine (Alum+CpG adjuvant combined with gHgL, A-gHgL) was obtained.

Comparative Example 2 Preparation of gB soluble vaccine

The preparation method of gB protein is the same as that of Example 3, except that gB is not linked to Ferritin protein via SpyCatcher-Spytag. Its amino acid sequence is shown in SEQ ID NO.22.

The preparation method of gB soluble vaccine was the same as that in Example 7, and 100 μL of gB soluble vaccine (Alum+CpG adjuvant combined with gB, A-gB) was obtained.

Comparative Example 3 Preparation of gp350ECD ₁₂₃ soluble vaccine

The preparation method of gp350ECD ₁₂₃ protein is the same as that of Example 4, except that gp350ECD ₁₂₃ is not linked to the Ferritin protein via SpyCatcher-Spytag, and its amino acid sequence is shown in SEQ ID NO.23.

The preparation method of gB soluble vaccine was the same as that of Example 8, and 100 μL of gp350ECD ₁₂₃ soluble vaccine (Alum+CpG adjuvant combined with gp350ECD ₁₂₃ , A-gp350ECD ₁₂₃ ) was obtained.

Comparative Example 4 Preparation of gp42 soluble vaccine

The preparation method of gp42 protein is the same as that of Example 5, except that gp42 is not linked to Ferritin protein via SpyCatcher-Spytag, and its amino acid sequence is shown in SEQ ID NO.24.

The preparation method of gp42 soluble vaccine was the same as that in Example 9, and 100 μL of gp42 soluble vaccine (Alum+CpG adjuvant combined with gp42, A-gp42) was obtained.

Effect Example 1 Nanoparticle Vaccine Particle Size and Morphology

1. Protein purification and assembly

The purification and assembly results of the components of the immunogenic complex prepared in Example 1-5 are shown in FIG1 . gHgL, gB, gp350ECD ₁₂₃ and gp42 were successfully linked to Ferritin to achieve multi-copy display, and almost no empty Ferritin particles existed after assembly.

2. Morphological characterization

The morphologies of Ferritin-gHgL, Ferritin-gB, Ferritin-gp350ECD ₁₂₃ and Ferritin-gp42 prepared in Examples 2-5 were observed using a transmission electron microscope.

As shown in Figure 2, the four immunogenic complexes have regular shapes and round appearances.

3. Particle size test and Zeta potential test:

The average particle sizes of Ferritin-gHgL, Ferritin-gB, Ferritin-gp350ECD ₁₂₃ and Ferritin-gp42 prepared in Examples 2-5 were tested using a Malvern particle size analyzer (with a dynamic light scattering detector).

As shown in Figure 3, the four nanoparticle vaccines all have a narrow and symmetrical particle size distribution.

Effect Example 2 Determination of the binding of immunogenic complexes and monomers with antigen-specific antibodies

1) SpyTag-Ferritin, Ferritin-gHgL, Ferritin-gB, Ferritin-gp350ECD ₁₂₃ , Ferritin-gp42, spycatcher-gHgL, spycatcher-gB, spycatcher-gp350ECD ₁₂₃ , spycatcher-gp42 prepared in Examples 1-5 and gHgL, gB, gp350ECD ₁₂₃ , gp42 prepared in Comparative Examples 1-4 were coated in a 96-well plate at 100 ng/well, 100 μL per well, and coated overnight at 4°C.

2) The plate coated overnight was washed once with PBST and blocked with blocking solution (20 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer solution with a pH value of 7.4 containing 20% calf serum and 1% casein) at 37°C for 2 h.

3) Dilute each antigen-specific antibody to 100 μg/ml in the first well, then dilute it 24 times in a doubling gradient (concentration range: 0.000001-1000000 ng/mL), add it to the wells coated with the antigen, and incubate at 37°C for 1 hour.

4) Wash 5 times, add 100 μl HRP-labeled secondary antibody of the corresponding species, and incubate at 37°C for 30 min.

5) Wash five times, add 100 μL TMB substrate to each well, incubate for 15 min in the dark, terminate the reaction with 50 μL 2M H ₂ SO ₄ , and detect OD values at 450 nm and 630 nm.

The results are shown in Figure 4 , and the binding ability of the nanoparticle vaccine to the antigen-specific monoclonal antibody is stronger than that of the monomer.

Effect Example 3 Evaluation of Nanoparticle Vaccine and Control Vaccine Activating Immune System in Mice

1. Immunization method

Female C57BL/6 mice aged 5 to 8 weeks were divided into ten groups A to Q, with 3 mice in each group. The mice were immunized by subcutaneous injection at the base of the tail according to the immunization scheme in Table 1, and booster immunizations were performed 2 weeks and 4 weeks after the first immunization, for a total of 3 immunizations.

Table 1 Immunoassay group design

2. Serum total antibody binding titer detection

On the 7th day after each immunization, blood was collected from the orbits of the mice, and the serum was separated and the IgG binding titer in the serum was detected by ELISA.

Testing process:

1) 100 ng/well of EBV recombinant protein antigen was coated in a 96-well plate, 100 μL per well, at 4°C overnight.

2) The plate coated overnight was washed once with PBST and blocked with blocking solution (20 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer solution with a pH value of 7.4 containing 20% calf serum and 1% casein) at 37°C for 2 h.

3) The immune serum or negative control serum was diluted 1:100 in the first well, and then diluted in multiples, added to the wells coated with antigen, and incubated at 37°C for 1 hour.

4) Wash 5 times, add 100 μl HRP-labeled goat anti-mouse secondary antibody, and incubate at 37°C for 30 min.

5) Wash five times, add 100 μL TMB substrate to each well, incubate for 15 min in the dark, terminate the reaction with 50 μL 2M H ₂ SO ₄ , and detect OD values at 450 nm and 630 nm.

The results of the total anti-binding titer test of the serum of each group of mice are shown in Figure 5A. The total anti-binding titer of the serum against each protein mediated by the nanoparticle vaccine after immunization was higher than that of the soluble protein vaccine, indicating the advantage of the nanoparticle vaccine in mediating humoral immune response. The serum binding titer after immunization with the nanoparticle vaccine was 14 to 43 times higher than that of the soluble protein vaccine. After three immunizations, the binding titers produced by the four nanoparticle vaccines A-F-gp350, A-F-gp42, A-F-gHgL and A-F-gB were (log10): 6.48, 6.41, 5.41, and 7.02, respectively, while the binding titers produced by the control soluble vaccines A-gp350, A-gp42, A-gHgL and A-gB were (log10): 5.34, 4.78, 4.23, and 5.64, respectively. The binding titers of the nanoparticle vaccines A-F-gp350, A-F-gp42, A-F-gHgL and A-F-gB after immunization increased by approximately 14, 43, 15, and 24 times, respectively, compared with the control group.

3. Serum neutralization titer detection

EBV B cell neutralization model: The serum was diluted with RPMI 1640 serum-free medium, 1:5 in the first well, 2-fold gradient dilution, and a total of 12 gradient dilutions; 20 μL of the diluted antibody was taken into a 96-well cell plate, and 20 μL of EBV-GFP virus suspension produced by CNE2 cells was added, mixed evenly, and placed in a 37°C incubator for incubation for 2 h. 1x10 ⁶ AKATA cells were resuspended in 16 mL 10% FBS RPMI1640 medium, 160 μl of the cell suspension was added to the virus and serum mixture, and the mixture was placed in a 37°C incubator for incubation for 48 hr. The virus infection rate of AKATA cells was detected using a flow cytometer LSRFortessaX-20 produced by BD. The reduction ratio of the number of GFP-positive cells in the antibody-treated group compared with the infection control group was calculated, and the inhibition rate (%) of the antibody in the B cell infection model was calculated. The inhibition rate and serum dilution multiple were plotted, and the half inhibition dilution multiple ID ₅₀ was obtained by fitting, which was the neutralization titer of the serum in the B cell model.

The results of B cell neutralization are shown in Figure 5B. Compared with the soluble protein vaccine, the serum after immunization with the four nanoparticles has a higher ability to neutralize B cell infection. The neutralization titers produced by the four nanoparticle vaccines A-F-gp350, A-F-gp42, A-F-gHgL and A-F-gB are (log10): 3.17, 3.61, 3.23, 3, respectively, while the neutralization titers produced by the control soluble vaccines A-gp350, A-gp42, A-gHgL and A-gB are (log10): 1.81, 2.12, 1.47, 1.73, respectively. The neutralization titers of the nanoparticle vaccines A-F-gp350, A-F-gp42, A-F-gHgL and A-F-gB after immunization were increased by about 23, 31, 58, and 19 times compared with the control group.

4. T cell response detection

1) On the seventh day after the third immunization, the spleen of the mice was taken and ground into a cell suspension.

2) Take 5x10 ⁶ cells from each well and stimulate with the corresponding antigen (10 μg). At the 12th hour of stimulation, inhibitors monensin and brefeldin A were added. At the 16th hour of stimulation, the cells were collected, washed three times with PBS, and blocked with anti-Fc at 4°C for 30 minutes.

3) Surface marker staining: anti-mouse APC/Cy7-CD45, anti-mouse FITC-CD3, anti-mouse AF700-CD4, anti-mouse APC-CD8, live and death, incubate at 4°C for 30 min, and wash three times with PBS.

3) After permeabilization of the cells using a membrane fixation kit, intracellular marker staining was performed: anti-mouse BV421-TNF-α, anti-mouse PC7-IFN-γ, anti-mouse PE-IL-2, incubated at room temperature for 30 minutes, washed three times, and detected by flow cytometry.

The results are shown in Figure 6A. After in vitro restimulation, the percentage of TNF-α-positive cells in the CD4 ⁺ T cells of the spleen of mice in the AF-gp350, AF-gp42, AF-gHgL and AF-gB groups were 1.16%, 1.15%, 1.86% and 1.30%, respectively, which were significantly higher than those in the corresponding free protein group (TNF-α ⁺ CD4 ⁺ T cell<1%); the percentage of TNF-α-positive cells in the CD8 ⁺ T cells of the spleen of mice in the AF-gp350, AF-gp42, AF-gHgL and AF-gB groups were 0.73%, 1.11%, 1.08% and 2.03%, respectively, which were significantly higher than those in the corresponding free protein group (TNF-α ⁺ CD8 ⁺ T cell<0.7%). Therefore, the nanoparticle vaccine can significantly activate the antigen-specific T cell immune response in mice after immunization. In general, the above results show that the four nanoparticle vaccines alone can induce efficient humoral and cellular immune responses in mice.

Effect Example 4 Evaluation of the immune memory mediated by nanoparticle vaccine and its control vaccine

1. Immunization method

The same effect as embodiment 3.

2. Evaluation of immune memory

1) On the seventh day after the third immunization, the spleen of the mice was taken and ground into a cell suspension.

2) Take 1x10 ⁵ cells, wash once with PBS, and then add antibodies: anti-mouse CD45-APC/Cy7, anti-mouse B220-BV605, anti-mouse CD8-APC, anti-mouse CD4-AF700, anti-mouse IgG-PerCP/Cy5.5, anti-mouse CD27-BV421, anti-mouse CD44-PE/Cy7, anti-mouse CD62L-PE, incubate at 4℃ for 30min

3) After washing three times with PBS, flow cytometry was performed.

As shown in Figure 7, the nanoparticle vaccine can effectively stimulate the production of immune memory. Among them, the number of CD4 effector T cells in the spleen of mice in the A-F-gp350, A-F-gp42, A-F-gHgL and A-F-gB groups was about 1.5, 1.6, 1.2, and 1.5 times higher than that of the corresponding soluble proteins, respectively; the number of CD8 effector T cells in the spleen of mice in the A-F-gp350, A-F-gp42, A-F-gHgL and A-F-gB groups was about 2.0, 2.2, 2.3, and 2.3 times higher than that of the corresponding soluble proteins, respectively; the number of memory B cells in the spleen of mice in the A-F-gp350, A-F-gp42, A-F-gHgL and A-F-gB groups was about 2.2, 2.1, 2.2, and 6.2 times higher than that of the corresponding soluble proteins, respectively.

Effect Example 5 Evaluation of the effect of nanoparticle vaccine combination

1. Immunization method

Female C57BL/6 mice aged 5 to 8 weeks were divided into two groups, A and B, with 3 mice in each group. The mice were immunized by subcutaneous injection at the base of the tail according to the immunization scheme in Table 2, and booster immunizations were performed 2 weeks and 4 weeks after the first immunization, for a total of 3 immunizations.

Table 2 Experimental design of nanoparticle combined vaccine

2. Serum neutralization titer detection

EBV B cell neutralization model: The serum was diluted with RPMI 1640 serum-free medium, 1:5 in the first well, 2-fold gradient dilution, and a total of 12 gradient dilutions; 20 μL of the diluted antibody was placed in a 96-well cell plate, and 20 μL of EBV-GFP virus suspension produced by CNE2 cells was added, mixed evenly, and placed in a 37°C incubator for incubation for 2 hours. 1x10 ⁶ AKATA cells were resuspended in 16 mL 10% FBS RPMI1640 medium, 160 μl of the cell suspension was added to the virus and serum mixture, and the mixture was placed in a 37°C incubator for incubation for 48 hr. The virus infection rate of AKATA cells was detected using a flow cytometer LSRFortessaX-20 produced by BD. The reduction ratio of the number of GFP-positive cells in the antibody-treated group compared with the infection control group was calculated, and the inhibition rate (%) of the antibody in the B cell infection model was calculated. The inhibition rate and serum dilution multiple were plotted, and the half inhibition dilution multiple ID ₅₀ was obtained by fitting, which was the neutralization titer of the serum in the B cell model.

The results of B cell neutralization are shown in Figure 8. When the nanoparticle vaccine is used in combination, the neutralization titer against B cells is about 11 times, 4 times, 9 times and 16 times higher than that of the single Ferritin-gp350ECD ₁₂₃ nanoparticle vaccine, Ferritin-gp42 nanoparticle vaccine, Ferritin-gHgL nanoparticle and Ferritin-gB nanoparticle, respectively. Therefore, compared with single nanoparticle immunization, the serum neutralization ability mediated by the combination of nanoparticles is greatly improved, and it is a more excellent candidate vaccine.

Claims (10)

1. An immunogenic complex comprising:

Recombinant protein a containing an antigen; and

Recombinant protein B containing a carrier protein;

the antigen is selected from any one of gHgL, gB, gp, gHgL, gB, gp and gp42 of EB virus;

the carrier protein is Ferritin;

The recombinant protein A and the recombinant protein B are covalently connected through SpyCatcher-SpyTag.

2. The immunogenic complex of claim 1, wherein:

The immunogenic complex is any one of an immunogenic complex A, an immunogenic complex B, an immunogenic complex C and an immunogenic complex D:

The immunocomplex a comprises:

Recombinant protein a containing an antigen; and

Recombinant protein B containing a carrier protein;

The antigen is gHgL of EB virus;

The immunocomplex B comprises:

Recombinant protein a containing an antigen; and

Recombinant protein B containing a carrier protein;

the antigen is gB of EB virus;

The immune complex C comprises:

Recombinant protein a containing an antigen; and

Recombinant protein B containing a carrier protein;

the antigen is gp350 of EB virus;

The immune complex D comprises:

Recombinant protein a containing an antigen; and

Recombinant protein B containing a carrier protein;

The antigen is gp42 of EB virus;

Preferably, the recombinant protein a comprises an antigen and SpyCatcher, and the recombinant protein B comprises a carrier protein and SpyTag;

Preferably, the recombinant protein a comprises an antigen and SpyTag and the recombinant protein B comprises a carrier protein and SpyCatcher.

3. The immunogenic complex of claim 2, wherein:

the antigen is connected with the SpyCatcher or the SpyTag through a connecting peptide; or (b)

The carrier protein is connected with the SpyCatcher or SpyTag through a connecting peptide;

Preferably, the amino acid sequence of the flexible connecting peptide is shown as SEQ ID NO. 3;

preferably, the recombinant protein a comprises the SpyCatcher, the connecting peptide and the antigen in order from the N-terminus to the C-terminus;

preferably, the recombinant protein B comprises the SpyTag, the connecting peptide and the carrier protein in order from the N-terminus to the C-terminus;

Preferably, the recombinant protein a further comprises a tag sequence;

Preferably, the recombinant protein B further comprises a tag sequence;

Preferably, the gHgL includes gL and gH; further comprises gL protein outer membrane region sequences 24-137 AA and gH protein outer membrane region sequences 19-678 AA, as shown in SEQ ID NO. 4;

Preferably, the gB comprises a gB protein ectodomain amino acid sequence 24-683 AA; further, amino acid 112-113 of gB is replaced by HR from WY, amino acid 193-196 is replaced by RVEA from WLIW, and the amino acid is shown in SEQ ID NO. 5;

Preferably, the gp350 is gp350 ECD ₁₂₃; further gp350 protein ECD123 region 2-425 AA, as shown in SEQ ID NO. 6;

preferably, gp42 is gp42 protein ectodomain sequence 34-223 AA, as shown in SEQ ID NO. 7;

Preferably, the amino acid sequence of the carrier protein Ferritin is shown in SEQ ID NO. 8;

preferably, the amino acid sequence of the SpyCatcher is shown as SEQ ID NO. 9;

Preferably, the amino acid sequence of the SpyTag is shown in SEQ ID NO. 10.

4. A biological material associated with the immunogenic complex of any one of claims 1-3, the biological material being any one of B1) to B12):

b1 A nucleic acid molecule encoding the immunogenic complex of any one of claims 1-3;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1);

b4 A recombinant vector comprising the expression cassette of B2);

b5 A recombinant microorganism comprising the nucleic acid molecule of B1);

B6 A recombinant microorganism comprising the expression cassette of B2);

B7 A recombinant microorganism containing the recombinant vector of B3);

B8 A recombinant microorganism comprising the recombinant vector of B4);

b9 A transgenic animal cell line comprising the nucleic acid molecule of B1);

b10 A transgenic animal cell line comprising the expression cassette of B2);

B11 A transgenic animal cell line comprising the recombinant vector of B3);

b12 A transgenic animal cell line comprising the recombinant vector of B4).

5. A method of preparing an immunogenic complex according to any one of claims 1 to 3, comprising incubating recombinant protein a and recombinant protein B to obtain;

Preferably, the molar mass ratio of the recombinant protein A to the recombinant protein B is (6-10): 1, a step of;

preferably, the incubation time is 4-24 hours;

preferably, the incubation temperature is 1-25 ℃.

6. The use of an immunogenic complex according to any one of claims 1 to 3, and/or a biomaterial according to claim 4 for the preparation of a medicament,

The drug has at least one function of c 1) to c 2):

c1 Prevention of EB virus infection;

c2 For treating and/or preventing diseases caused by EB virus infection.

7. A medicament, comprising: the immunogenic complex of any one of claims 1-3; and/or

The biomaterial of claim 4;

Preferably, the medicament further comprises pharmaceutically acceptable excipients.

Any one of vaccine a 1) to a 2):

a1 A vaccine comprising: the immunogenic complex of any one of claims 1-3; and/or

The biomaterial of claim 4;

a2 A vaccine comprising: at least two of the immunogenic complex a, the immunogenic complex B, the immunogenic complex C, the immunogenic complex D of any one of claims 2-3.

9. The vaccine of claim 8, wherein:

a2 In which vaccine comprises an immunogenic complex a, an immunogenic complex B, an immunogenic complex C, and an immunogenic complex D as described in any one of claims 2-3;

preferably, the vaccine of a 1), a 2) further comprises: an adjuvant.

10. Kit of parts comprising a vaccine according to claim 9 or 10 and a container for said vaccination.