Chimeric design of ebv antigens and uses thereof

Abstract

The application discloses an EB virus antigen of a chimeric design and application thereof. The EB virus chimeric antigen provided by the application is rationally designed based on structural biology, and gL, gH and gp42 proteins of an EB virus are connected through a linker. The antigen of the chimeric design simultaneously stably presents antigen epitopes of the three proteins, can increase the immunogenicity of a vaccine, and can simplify a production amplification process and quality control. The EB virus chimeric antigen can be used alone or in combination with other antigens, has high immunogenicity when used as a vaccine or a vaccine component, can induce an immune animal to produce a high level of neutralizing antibodies, can be used for preparing a vaccine for preventing or treating EB virus infection, and can be used as a detection reagent for the EB virus.

Description

Technical Field

[0001] This invention relates to a chimeric EB virus antigen and its application, belonging to the field of biomedical technology. Background Technology

[0002] Epstein-Barr virus (EBV) is one of the most widespread viruses in the world. It is an infectious and carcinogenic human herpesvirus. EBV infection and latency are associated with various lymphomas, epithelial cancers, and non-malignant diseases, such as multiple lymphoproliferative disorders, B / T / NK cell lymphoma, nasopharyngeal carcinoma (NPC), gastric cancer (GC), infectious mononucleosis (IM), oral hairy leukoplakia, systemic lupus erythematosus, and multiple sclerosis (MS). The development of EBV vaccines is crucial for the prevention, control, and treatment of EBV-related diseases. However, due to the complex life cycle of EBV, unclear infection and immune mechanisms, and limitations of animal models, no effective EBV vaccine has yet been approved for marketing.

[0003] EBV was first discovered in Burkitt lymphoma (BL) in 1964. Belonging to the subfamily Gammaherpesvirinae of the family Herpesviridae, it is an enveloped double-stranded DNA virus with a diameter of 150–200 nm. EBV has the characteristic three-layered structure of herpesviruses: ① an outer lipid bilayer envelope containing viral glycoproteins responsible for host recognition and membrane fusion; ② an intermediate envelope containing 20–40 different viral proteins; ③ an icosahedral nucleocapsid composed of 162 capsomeres, containing a 172 kb double-stranded DNA genome.

[0004] The EBV genome encodes more than 80 genes, including structural proteins, latent phase proteins, and lysis phase proteins. Among these, gp350, gB, gH, gL, and gp42 are structural proteins. EBV is mainly transmitted through saliva, crossing the mucosal epithelial cell barrier via membrane fusion, and then infecting B cells in the secondary metastatic lymphoid tissue of the mucosa (endocytosis pathway). A study of the contribution of different antibodies in the serum of >1000 healthy individuals to the neutralization of EBV infection showed that for B cell neutralization, antibodies against gp350, gH / gL, and gp42 accounted for 44.6% ± 4.37%, 46.9% ± 3.29%, and 10.9% ± 1.85%, respectively, with the additional neutralizing activity in B cells likely provided by gB antibodies. For epithelial cell neutralization, gH / gL antibodies contributed 76.0% ± 0.89%, with the additional neutralizing activity likely provided by gB or BMRF2 antibodies. Therefore, gp350, gH / gL and gp42 antibodies in human plasma constitute most of the neutralizing activity against B cell infection, while gH / gL antibody is the main component that inhibits epithelial cell infection.

[0005] Current research focuses primarily on vaccines designed targeting the viral envelope glycoprotein gp350. While gp350 was the earliest discovered neutralizing target, it failed to prevent EBV infection in clinical trials. In recent years, with increased research into EBV infection and immune mechanisms, researchers have attempted to incorporate four envelope glycoproteins involved in viral invasion—gH, gL, gB, and gp42—into vaccine design as targets for prophylactic vaccines. Multi-component vaccines are expected to elicit higher neutralizing antibody titers compared to single-component vaccines. However, the production and quality control of multi-component vaccines are typically more complex. Therefore, this invention aims to rationally design multi-component vaccines using structural biology techniques. Through rational sequence arrangement, linker connection, and the introduction of disulfide bond mutations, the goal is to obtain a chimeric antigen covering multiple component epitopes. This would maintain the immunogenicity of the original multi-component antigen while simplifying production and quality control processes, thus facilitating the industrialization of vaccine products. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a chimeric EB virus antigen in which gL, gH, and gp42 are linked together in a specific sequential order via linkers, enabling the simultaneous and stable presentation of the three antigenic epitopes. Animal experiments have shown that high-titer neutralizing antibodies are generated after immunization, providing a new direction for clinical applications and laying the foundation for developing new products to prevent and block EB infection.

[0007] This invention focuses on the design of EBV chimeric antigens. Guided by structural biology and bioinformatics, it designs specific chimeric antigens that stably present multi-component antigenic epitopes. These chimeric antigens link the gL, gH, and gp42 proteins of EBV via linkers. This chimeric design allows for the simultaneous and stable presentation of epitopes from all three proteins, simplifying production scale-up processes and quality control while increasing vaccine immunogenicity. The EBV chimeric antigens enable large-scale soluble expression of the target proteins in mammalian cells, exhibiting excellent protein stability. When used as a vaccine or vaccine component, its immunogenicity is enhanced, inducing high levels of neutralizing antibodies in immunized animals. The EBV chimeric antigens described in this application can be used for the prevention and / or treatment of EBV infection, and can also be used with EBV detection reagents. When used for the prevention and / or treatment of EBV infection, the chimeric antigens can be used alone, in combination with other EBV antigens, in vaccine compositions with different adjuvants, or in multivalent vaccines with different types of vaccine products.

[0008] The first objective of this invention is to provide a chimeric EB virus antigen, wherein the chimeric antigen is formed by linking the three structural proteins of EB virus, gL, gH and gp42, in sequence using linkers to form a whole.

[0009] Furthermore, the gL structural protein is the soluble expression region of the gL protein with the amino acid sequence shown in SEQ ID NO.1, or a protein with more than 95% homology and the same or substantially the same immunogenicity.

[0010] Furthermore, the amino acid sequence of the gL structural protein is the amino acid sequence from position 23 to position 137 as shown in SEQ ID NO.1, or a further truncated amino acid sequence.

[0011] Furthermore, the amino acid sequence of the gL structural protein is the amino acid sequence from position 24 to position 135 as shown in SEQ ID NO.1.

[0012] Furthermore, the gH structural protein is the soluble expression region of the gH protein with the amino acid sequence shown in SEQ ID NO.2, or a protein with more than 95% homology and the same or substantially the same immunogenicity.

[0013] Furthermore, the amino acid sequence of the gH structural protein is the amino acid sequence from position 18 to position 679 as shown in SEQ ID NO.2, or a further truncated amino acid sequence.

[0014] Furthermore, the amino acid sequence of the gH structural protein is the sequence of amino acids from position 20 to position 675 as shown in SEQ ID NO.2.

[0015] Furthermore, the gp42 structural protein is the soluble expression region of the gp42 protein with the amino acid sequence shown in SEQ ID NO.3, or a protein with more than 95% homology and the same or substantially the same immunogenicity.

[0016] Furthermore, the amino acid sequence of the gp42 structural protein is the amino acid sequence from position 57 to position 251 as shown in SEQ ID NO.3, or a further truncated amino acid sequence.

[0017] Furthermore, the amino acid sequence of the gp42 structural protein is the amino acid sequence from position 75 to position 251 as shown in SEQ ID NO.3.

[0018] Furthermore, the linker is a fragment of 1-30 amino acids.

[0019] In this invention, a conventional connector structure can be selected.

[0020] Furthermore, the structure of the connector is (GGS)n, where n is an integer between 1 and 10.

[0021] Specifically, in some embodiments, GGGGSGGGG connectors are used between gL and gH, and GGSGG connectors are used between gH and GP42.

[0022] Specifically, in some embodiments, an enzymatically cleavable GRSRR linker is used between gL and gH, and a GRSRRSRGGS linker is used between gH and GP42.

[0023] In this invention, when an enzymatically cleavable linker is used, the prepared chimeric antigen can be expressed normally, and the monomeric protein can be obtained by subsequent enzymatic digestion.

[0024] Furthermore, the chimeric antigen may also include at least one disulfide bond mutation.

[0025] In this invention, the structural stability of the chimeric antigen can be increased by adding one or more disulfide bond mutations.

[0026] Furthermore, the disulfide bond is mutated into a disulfide bond between the three structural proteins gL, gH, and gp42, or a disulfide bond within a structural protein.

[0027] Specifically, in some embodiments, the disulfide bond mutation is to mutate N84 in the gL sequence and G209 in the gH sequence to cysteine ​​C.

[0028] In this invention, the design of the gL-gH-gp42-DS mutant is mainly achieved by introducing a pair of disulfide bond mutations and replacing the linker with an enzymatically cleavable linker. After these two modifications, the antigen can still be expressed normally to prepare an antigen containing three components, indicating that the modification has a certain degree of tolerance.

[0029] Furthermore, the chimeric antigen can also be used in combination with other antigens to further increase the immunogenicity of the antigen.

[0030] Specifically, in some embodiments, the chimeric antigen may be used in combination with the gB protein of EB virus to increase immunogenicity.

[0031] A second objective of this invention is to provide a nucleic acid molecule encoding the EB virus chimeric antigen.

[0032] Furthermore, the nucleic acid molecule has been codon-optimized for expression in mammalian cells.

[0033] A third object of the present invention is to provide a carrier that contains the nucleic acid molecule.

[0034] A fourth object of the present invention is to provide a cell that expresses the EB virus chimeric antigen, or contains the nucleic acid molecule, or contains the vector.

[0035] A fifth object of the present invention is to provide a vaccine comprising the EB virus chimeric antigen.

[0036] Furthermore, the vaccine also contains other active ingredients.

[0037] Furthermore, the vaccine also contains a vaccine adjuvant.

[0038] A sixth object of the present invention is to provide a pharmaceutical composition comprising the EB virus chimeric antigen, the vaccine, the nucleic acid molecule, or the carrier.

[0039] Furthermore, the pharmaceutical composition includes pharmaceutically acceptable excipients.

[0040] Furthermore, the pharmaceutically acceptable excipients include protectants, stabilizers, preservatives, bactericides, inactivators, adjuvants, and / or buffers.

[0041] A seventh object of the present invention is to provide the use of the EB virus chimeric antigen, the nucleic acid molecule, the vector, the cell, the vaccine, or the pharmaceutical composition in the preparation of products for detecting, preventing, and / or treating EB virus infection.

[0042] By means of the above-described solution, the present invention has at least the following advantages:

[0043] This application provides a chimeric EB virus antigen. Based on structural biology techniques, a multi-component vaccine is rationally designed to obtain a chimeric antigen covering multiple component epitopes. By using linkers to sequentially connect the three proteins of EB virus (gL, gH, and gp42) to form a whole, the immunogenicity of the original multi-component antigen is maintained while simplifying the production and quality control process, which is conducive to the industrialization of vaccine products. Furthermore, the multi-component chimeric vaccine design of this application can elicit superior neutralizing antibody titers compared to single-component vaccines, providing a new direction for clinical applications and laying the foundation for developing new products to prevent and block EB infection.

[0044] The above description is only an overview of the technical solution and some results of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described below in conjunction with detailed drawings. Attached Figure Description

[0045] Figure 1Analysis of different purified proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing (R) and non-reducing (NR) conditions;

[0046] Figure 2 SEC analysis of chimeric antigen gL-gH-gp42;

[0047] Figure 3 Immunogenicity (binding antibodies and neutralizing antibodies) assessment results of vaccine compositions prepared from different components. Detailed Implementation

[0048] This invention discloses a chimeric EB virus antigen and its application. Those skilled in the art can refer to the content of this document and make appropriate improvements to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0049] the term:

[0050] In this application, the term "EB virus" or "EBV" belongs to the subfamily Gammaherpesvirus of the family Herpesviridae. Infection and latency of the virus are associated with a variety of lymphomas, epithelial cancers, and non-malignant diseases, such as multiple lymphoproliferative disorders, B / T / NK cell lymphoma, nasopharyngeal carcinoma (NPC), gastric cancer (GC), infectious mononucleosis (IM), oral hairy leukoplakia, systemic lupus erythematosus, and multiple sclerosis (MS).

[0051] In this application, the term "chimeric antigen" generally refers to the tandem expression of multiple antigens via linkers, where a single sequence contains multiple antigenic epitopes, and a target protein exhibiting multiple antigenic epitopes can be obtained through a single recombinant expression. When used as a vaccine component, this can significantly increase immunogenicity and protection. In this application, chimeric antigen refers to a design that links EB virus gL, gH, and gp42 antigens together via linkers.

[0052] In this application, the term "amino acid mutation" generally refers to a modification of an amino acid in the parental amino acid sequence. For example, the modification may include the substitution, insertion, and / or deletion of one or more amino acids. In this application, the amino acid mutation may include the deletion or substitution of at least one amino acid residue at a specified position in the amino acid sequence. In some embodiments, the amino acid mutation can optimize the conformation of the protein constituted by the amino acid sequence. Amino acid mutations can be generated using genetic or chemical methods well known in the art. For example, genetic methods may include site-directed mutagenesis, PCR, and gene synthesis.

[0053] In this application, the term "codon optimization" generally refers to improving the expression of the nucleic acid encoding the polypeptide by replacing one or more codons of the same amino acid residue encoded by the parent polypeptide with codons that have different relative frequencies of use. In this application, all possible codons encoding the mutated amino acid are within the scope of protection of this application, provided that the amino acid mutation is the same as that in this application.

[0054] In this application, the term "signal peptide" generally refers to an amino acid sequence present at the N-terminus of a transmembrane protein as a signal during transmembrane transport. For example, the transmembrane protein may include secretory proteins or cell membrane proteins. For example, the signal peptide may be synthesized at the N-terminus of the transmembrane protein in the form of a precursor polypeptide.

[0055] In this application, the description of protein mutation sites is typically expressed as "amino acid + amino acid position + mutated amino acid". In this application, the mutation may include, but is not limited to, the addition, substitution, deletion, and / or removal of amino acids. For example, the term "N84C" typically refers to the mutation of asparagine (N) at position 84 to cysteine ​​(C).

[0056] In this application, the term "nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide, or ribonucleotide of any length in an isolated form, or an analogue isolated from its natural environment or synthesized artificially.

[0057] In this application, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted, thereby enabling the protein to be expressed. Vectors can be used to transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cells. Examples of vectors include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that assist them in entering cells or in integrating the target element into the host cell, such as viral particles, liposomes, protein coats, or integrase, but not only these substances.

[0058] In this application, the term "pharmaceutical composition" generally refers to a composition for the prevention / treatment of a disease or condition. The pharmaceutical composition may comprise the EB virus chimeric antigen described in this application, the nucleic acid molecule described in this application, the vector described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are generally non-toxic to the recipient at the doses and concentrations used.

[0059] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) peptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming anti-charge ions such as sodium, and / or nonionic surfactants.

[0060] In this application, the term "adjuvant" generally refers to any substance that assists or modulates the action of a drug, including but not limited to immunological adjuvants, which enhance or diversify the immune response to an antigen. In this application, the adjuvant can be used to enhance the antigenicity of the EB virus antigen. In some embodiments, the adjuvant may comprise a mineral suspension (e.g., alum, aluminum hydroxide, or phosphate). In some embodiments, the adjuvant may comprise an oil-in-water emulsion. In some embodiments, the adjuvant may comprise liposomes. In some embodiments, the adjuvant may comprise immunostimulants such as MPL, CpG, and Poly:IC.

[0061] In this application, the term "vaccine" refers to a pharmaceutical composition comprising an immunogen capable of evoking a preventive or therapeutic immune response in an individual. Typically, vaccines elicit an antigen-specific immune response against a pathogen, such as a viral pathogen.

[0062] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0063] This application, based on structural biology and combined with immunological principles, designs a novel protective vaccine with high safety, broad-spectrum protection, and reliable preparation process. The EB chimeric design antigen provided in this application stably presents antigenic epitopes of three proteins simultaneously, which can increase vaccine immunogenicity while simplifying the production scale-up process and quality control. The EB virus chimeric antigen described in this invention can be used alone or in combination with other antigens. When used as a vaccine or vaccine component, it exhibits high immunogenicity, inducing high levels of neutralizing antibodies in immunized animals. It can be used to prepare vaccines for the prevention or treatment of EB virus infection, and can also be used as a reagent for the detection of EB virus.

[0064] Without being limited by any theory, the embodiments described below are merely for illustrating the EB virus chimeric antigen, preparation method, quality characterization and use of this application, and are not intended to limit the scope of the invention.

[0065] The present invention will be further illustrated below with reference to the embodiments:

[0066] Example 1: Design of EBV protein mutants

[0067] This invention relates to the design and evaluation of various EBV protein mutants. This embodiment summarizes five typical examples, including gp42 expressed alone, gH-gL expressed in a two-component chimeric structure of gH and gL, gp42 and gL-gH-gp42 expressed in a three-component chimeric structure of gH and gL, the three-component chimeric antigen gL-gH-gp42-DS with disulfide bonds and an enzyme-cleavable linker, and gB protein mutant expressed alone.

[0068] The gp42 expressed alone was referenced from the published sequence in GenBank:XOL08407.1. The amino acid sequence of gp42 in the range of G62-S251 as shown in SEQ ID NO.3 was selected. A signal peptide sequence derived from gH (MQLLCVFCLVLLWEVGA) was added to the N-terminus, and a protein tag of 6 histidines was added to the C-terminus.

[0069] The gH-gL chimeric antigen connects gH and gL via a GS linker. The gH portion is selected from the amino acid sequence in the range of M1-A678 in the gH sequence shown in SEQ ID NO.2; the gL portion is selected from the amino acid sequence in the range of W24-G137 in the gL sequence shown in SEQ ID NO.1. The gL portion is connected to the C-terminus of the gH portion using a GS linker (GGGGSGGGGSGGGGS).

[0070] In designing the three-component chimeric antigen (gp42, gH, and gL), various different ligation sequences were attempted, such as directly linking gp42 to the C-terminus of the gH-gL chimeric antigen via a linker. However, no soluble target protein was obtained. In this embodiment, the preferred three-component chimeric antigen, based on structural biology analysis, adopts the gL-gH-gp42 ligation sequence. The gL portion is selected from the amino acid sequence W24-R135 of SEQ ID NO.1, the gH portion from the amino acid sequence A18-A678 of SEQ ID NO.2, and the gp42 portion from the amino acid sequence R64-S251 of SEQ ID NO.3. The fusion is performed from the N-terminus to the C-terminus using the gL-gH-gp42 sequence, with the gL-gH linker using GGGGSGGGG linker and the gH-gp42 linker using GGSGG linker.

[0071] Based on the design of the gL-gH-gp42 chimeric antigen, the gL-gH-gp42-DS mutant further investigated the effects of introducing disulfide bonds and cleavable sites on the chimeric antigen. The gL portion was selected from the amino acid sequence W24-H134 of SEQ ID NO.1, the gH portion from the amino acid sequence L20-E675 of SEQ ID NO.2, and the gp42 portion from the amino acid sequence K75-S251 of SEQ ID NO.3. Fusion was performed from the N-terminus to the C-terminus using the gL-gH-gpP42 sequence, with a cleavable GRSRR linker between gL and gH, and a GRSRRSRSGGS linker between gH and gp42. To further increase the stability of the gL and gH portions after cleavage, N84 in the gL sequence and G209 in the gH sequence were mutated to cysteine ​​C.

[0072] The gB mutant was developed using the publicly available sequence YP_401713.1 from the NCBI database as a reference. The amino acid sequence within the range of M1-D691 was selected, and the GGSGGS linker and the Foldon trimer domain of the T4 phage containing the His tag were added to the C-terminus. The hydrophobic peptide W193-W196 in the fusion peptide was replaced with the hydrophilic RVEA peptide to improve protein solubility. The potential furin restriction sites R429-R431 were mutated to GSG to avoid cleavage, and the S467 site was mutated to proline P and the R662-Y664 sites were mutated to phenylalanine-cysteine-cysteine ​​(FCC) residues to introduce disulfide bonds between trimers to stabilize the trimer structure of the gB protein.

[0073] The amino acid sequence of the aforementioned protein mutant was codon optimized by General Biotechnology Co., Ltd. using CHO as the host. HindIII and kozark sequences (AAGCTTGCCGCCACC) were added to the 5' end of the gene, and a stop codon and NotI restriction site (TGATAAGCGGCCGC) were added to the 3' end before gene synthesis. The synthesized gene was cloned into the pKS001 mammalian cell vector (Zhongshan Kangsheng, A13201) containing the CMV promoter and SV40 polyA, and plasmids for transfection were extracted for subsequent cell transfection and protein expression testing.

[0074] Example 2: Expression of EBV protein mutants

[0075] HEK293F cells, characterized by rapid growth and good cell morphology, were used as the transfection host for small-volume transfection. Before transfection, the viable cell density of the seed cells was adjusted to 6 million cells per milliliter, with 20 mL of cell culture used for each project. For each project, 20 μg of plasmid and 100 μg of PEI MAX transfection reagent (manufacturer: Polysciences, catalog number: 23966-1) were diluted and mixed with 600 μL of OPM-293CD05 medium (manufacturer: OPMI, catalog number: 81075-001). The diluted transfection reagent was then slowly added to the diluted plasmid and gently mixed. The mixture was incubated at room temperature for 8 minutes to prepare the plasmid complex. After incubation, the prepared plasmid complex was slowly added to the cell culture medium to be transfected, and the cell culture was placed in a 37°C, 8% CO2 shaking incubator at 200 rpm. 18-24 hours after transfection, sample and monitor cell status, and add 2ml of BalanCD CHO Feed 4 (0.8X) (manufacturer: Fujifilm, catalog number: 94134) to each flask of transfected cells. End culture on the 4th day after transfection, and transfer the cell culture supernatant to the purification section for purification.

[0076] Example 3: Isolation and purification of EBV recombinant proteins with different molecular designs

[0077] To obtain high-purity gB, gp42, gH-gL, gH-gL-gp42, and gH-gL-gp42-DS recombinant proteins, culture harvests of different molecularly designed EBV proteins expressed in HEK293F cells were isolated and purified. A two-step method was selected for isolation and purification, including a crude purification capture step and a size exclusion chromatography purification step.

[0078] Molecules crudely purified using affinity chromatography (e.g., gB, gp42, gH-gL-gp42) containing affinity tags in their designed sequences were purified by diluting the culture harvest medium 1:1 with 1xPBS pH 7.4. The diluted protein was then captured using a Polar MC30-Ni Excel (Sepax 270630800) column pre-equilibrated with 1xPBS pH 7.4. After capture, the protein was eluted with 1xPBS pH 7.4 containing 500 mmol / L imidazole to obtain crude purified protein. Alternatively, a HiTrap column pre-equilibrated with 1xPBS pH 7.4 could be used. TM ProteinSelect TM (Cytiva17542152) The protein was captured by a chromatography column, held on the column, and eluted with 1xPBS at pH 7.4 to obtain crude purified protein.

[0079] Crudely purified molecules (such as gH-gL, gH-gL-gp42-DS) were obtained using cation exchange chromatography. The designed sequences did not contain any tags. After adjusting the pH with dilute hydrochloric acid and the conductivity with 20 mmol / L phosphate buffer, the molecules were captured using a CEX (UniGel 80SP) (Nano Micro 04082-080100-2050) chromatography column pre-equilibrated with 20 mmol / L phosphate buffer. After capture, the molecules were eluted with 20 mmol / L phosphate buffer containing 1 mol / L NaCl to obtain the crudely purified protein.

[0080] All crudely purified proteins were further purified using size exclusion chromatography (SLC), with either HiLoad 16 / 600 Superdex 200 pg (Cytiva 28989335) or Superdex 200 Increase 10 / 300 GL (Cytiva 28990944) selected based on the concentration. Before loading, the column system was equilibrated with 1xPBS at pH 7.4 until baseline stability was achieved. Then, the concentrated crudely purified protein sample was added using a syringe. After products at different retention times eluted, the target protein fraction was collected to obtain high-purity EBV recombinant protein.

[0081] like Figure 1 As shown, high-purity target proteins were obtained from EBV recombinant proteins with different molecular designs after separation and purification.

[0082] Example 4: Particle size distribution analysis of the designed chimeric EB antigen

[0083] To analyze the size distribution of the designed EB chimeric antigen, this experiment employed SEC-HPLC to analyze the purified sample. The specific procedure was as follows: the chromatographic column was a NanoChrom BioCore SEC-300 (5μm). A 7.8*300mm column was used, with a mobile phase of 20mM PB + 300mM NaCl at pH 7.4, a flow rate of 0.5ml / min, and a detection wavelength of 280nm. Isocratic elution was performed. Separation and detection were performed using a Shimadzu LC-20A system, with proteins eluted according to the molecular size of the analytes.

[0084] like Figure 2 As shown, the designed chimeric antigen has a single peak position and symmetrical peak shape, exhibiting a good uniform conformation.

[0085] Example 5: Immune response to chimeric EB antigen in mice

[0086] To compare the immunogenicity of immunization with different EBV protein components, either alone or in combination, 5-6 week old female BALB / c mice were randomly assigned to seven groups of six mice each: gB, gp42, gH-gL, gL-gH-gp42, gH-gL+gp42, gB+gL-gH-gp42 antigen groups, and a saline control group. Immunization was administered intramuscularly at 50 μl per mouse on days 0 and 21. Serum was collected on day 42 for the determination of binding and neutralizing antibody titers.

[0087] Table 1 Prescription Information

[0088]

[0089]

[0090] The specific preparation method for the vaccine composition is as follows: First, adsorb CpG7909 (Guangzhou Ruibo Biotechnology Co., Ltd.) onto aluminum hydroxide adjuvant (CRODA, catalog number AJV3012) (the content of aluminum hydroxide adjuvant here is essentially the content of aluminum element). Then, add the antigen to the CpG7909 / aluminum hydroxide sample, and use it after adsorption at room temperature for 30 minutes. If not used immediately, store at 2-8℃.

[0091] The antibody titer detection method was as follows: Indirect ELISA was used to detect the antibody titer. The antigen was diluted to 2 μg / ml with buffer, and 100 μl / well was coated onto a 96-well plate and incubated overnight at 2–8°C. After washing with PBST, blocking buffer (PBST containing 5% skim milk powder) was added at 300 μl / well, and incubated at 25°C for 1 hour. After washing with PBST, serum test sample (diluted to a certain factor with blocking buffer, then serially diluted 2-fold for a total of 11 dilutions, with replicates, the initial dilution for the saline group being 100) and blank control (blocking buffer, 4 wells per plate) were added at 100 μl / well, and incubated at 25°C. Incubate for 2 hours; after washing, incubate with HRP-labeled secondary antibody (BETHYL, A90-116P, diluted 5000 times with blocking buffer), 100 μl / well, at 25℃ for 1 hour; finally, add TMB chromogenic solution, 100 μl / well, and incubate at 25℃ for 10 minutes. After the reaction is terminated, read the absorbance value at 450 nm in a microplate reader. The cut-off value for this experiment is set at 2.1 times the average absorbance of the blank control in the same day's experiment. Data screening and analysis are performed using GraphPad Prism 8.0 software.

[0092] The detection method for neutralizing antibody titers is as follows: (1) Serum gradient dilution: In a V-bottom 96-well plate, the first well is diluted 1:10 (serum-free RPMI1640 is diluted, and the actual first well dilution factor after adding the virus is 1:20), and serum-free RPMI1640 is diluted three times in 8 gradients (20 μl). (2) Add 20 μl of CNE2-AKATA-EBV-GFP virus (infection efficiency ~28.45%) to the above serum dilution wells and incubate at 37℃ for 2 hours. Positive control: 20 μl of serum-free RPMI1640 (without serum) + 20 μl of virus. (3) Akata-neg plating: Akata-neg cells are plated into a 96-well plate at a density of 10,000 cells / well / 160 μl; (4) Add 40 μl of the above virus + serum mixture (total 200 μl) to the cell culture wells after incubation for 2 hours and culture for 48 hours. (5) Collect cells from each well individually into EP tubes and detect the GFP positivity rate by flow cytometry. (6) Fitting: GraphPad Prism four-parameter nonlinear fitting was used to calculate ID50.

[0093] like Figure 3 As shown, after two doses of immunization, compared with the saline group, the binding antibody levels of each antigen group were significantly increased, and the differences between vaccine groups were small. However, the neutralizing antibody titer levels differed significantly between groups. After the second immunization, the neutralizing antibody titers of the multi-component antigen group were higher than those of the single-component antigen group. Among the multi-component antigen groups, the two groups containing the gL-gH-gp42 tri-component antigen had the highest neutralizing antibody titers. The neutralizing antibody titer level of the gL-gH-gp42 chimeric antigen in this application was comparable to that of the antigens used after the single components were expressed separately and then combined.

[0094] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A chimeric EB virus antigen, characterized in that, The chimeric antigen is formed by linking the three structural proteins of EB virus, gL, gH, and gp42, in sequence using linkers to form a whole. The amino acid sequence of the gL structural protein is amino acid sequence from position 24 to position 135 as shown in SEQ ID NO.1, the amino acid sequence of the gH structural protein is amino acid sequence from position 18 to position 678 as shown in SEQ ID NO.2, and the amino acid sequence of the gp42 structural protein is amino acid sequence from position 64 to position 251 as shown in SEQ ID NO.3; the gL-gH are linked by the GGGGSGGGG linker, and the gH-GP42 are linked by the GGSGG linker.

2. A nucleic acid molecule encoding the EB virus chimeric antigen of claim 1.

3. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 2.

4. A cell, characterized in that, The cell expresses the EB virus chimeric antigen of claim 1, or contains the nucleic acid molecule of claim 2, or contains the vector of claim 3.

5. A vaccine, characterized in that, The vaccine contains the EB virus chimeric antigen as described in claim 1.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the EB virus chimeric antigen of claim 1, the vaccine of claim 5, the nucleic acid molecule of claim 2, or the vector of claim 3.

7. The use of the EB virus chimeric antigen of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, the cell of claim 4, the vaccine of claim 5, or the pharmaceutical composition of claim 6 in the preparation of products for the detection, prevention, and / or treatment of EB virus infection.

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