Chimeric EB virus antigen and application thereof

Abstract

The invention discloses an EB (Epstein-Barr) virus antigen with chimeric design and application thereof. The EB virus chimeric antigen provided by the invention is reasonably designed on the basis of structural biology, gL, gH and gp42 proteins of the EB virus are connected through a linker, and the chimeric designed antigen stably presents antigen epitopes of the three proteins at the same time, so that the production amplification process and quality control can be simplified while the vaccine immunogenicity is improved. The EB virus chimeric antigen can be used independently or in combination with other antigens, has high immunogenicity when being used as a vaccine or a vaccine component, can induce an immunized animal to generate a high-level neutralizing antibody, can be used for preparing a vaccine for preventing or treating EB virus infection, and can also be used as a reagent for detecting EB virus.

Description

Technical Field

[0001] The present invention relates to a chimeric-designed Epstein-Barr virus antigen and its application, belonging to the field of biomedical technology. Background Art

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

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

[0004] The genome of EBV encodes more than 80 genes, including structural proteins, latency proteins, lytic proteins, etc. Among them, gp350, gB, gH, gL, gp42, etc. are structural proteins. EBV is mainly transmitted through saliva. It crosses the mucosal epithelial cell barrier through the membrane fusion pathway and then infects B cells in the secondary lymphoid tissue under the mucosa (endocytic pathway). A study on the contribution ratio of different antibodies in the sera of >1000 healthy people to neutralize EBV infection showed that for the neutralization of B cells, the antibodies of gp350, gH / gL, and gp42 accounted for 44.6% ± 4.37%, 46.9% ± 3.29%, and 10.9% ± 1.85% respectively. The additional neutralizing activity in B cells may be provided by the antibodies of gB; while for the neutralization of epithelial cells, the antibodies of gH / gL contributed 76.0% ± 0.89%, and the additional neutralizing activity may be provided by the antibodies of gB or BMRF2. Therefore, the antibodies of gp350, gH / gL, and gp42 in human plasma constitute most of the neutralizing activity to prevent B cell infection, and the gH / gL antibody is the main component to inhibit epithelial cell infection.

[0005] Currently, more research has been focused on vaccines designed against the viral envelope glycoprotein gp350, which was the first neutralization target discovered but failed to prevent EBV infection in clinical trials. In recent years, with the research on EBV infection and immune mechanisms, researchers have attempted to incorporate the four envelope glycoproteins involved in viral invasion, namely gH, gL, gB, and gp42, into vaccine design as targets for preventive vaccines. The design of multi-component vaccines is expected to elicit better neutralizing antibody titers compared to single-component vaccines. However, the production, quality control, etc. of multi-component vaccines are usually more complex. Therefore, the present invention aims to rationally design multi-component vaccines based on structural biology techniques. Through reasonable sequence arrangement, linker connection, introduction of disulfide bond mutations, etc., it is hoped to obtain a chimeric antigen that covers multiple component epitopes, simplifies the production and quality control processes while maintaining the immunogenicity of the original multi-component antigen, and contributes to the industrialization of vaccine products. Summary of the Invention

[0006] To solve the above problems, the present invention provides a chimerically designed Epstein - Barr virus antigen. This antigen connects gL, gH, and gp42 in a specific order through a linker, and can simultaneously and stably present the epitopes of the three components. Animal experiments show that high - titer neutralizing antibodies can be produced after immunization of the body, providing a new direction for clinical application and laying a foundation for the development of new products for preventing and blocking EBV infection.

[0007] The present invention focuses on the design of Epstein - Barr virus chimeric antigens. Guided by structural biology and bioinformatics, a chimeric antigen that can stably present multi - component epitopes is designed. The chimeric antigen connects the gL, gH, and gp42 proteins of Epstein - Barr virus through a linker. This chimerically designed antigen can simultaneously and stably present the epitopes of the three proteins, can simplify the production scale - up process and quality control while increasing the immunogenicity of the vaccine. The Epstein - Barr virus chimeric antigen can be expressed in a large - scale soluble form in mammalian cells with excellent protein stability. When used as a vaccine or a vaccine component, its immunogenicity is enhanced, and it can induce a higher level of neutralizing antibodies in immunized animals. The Epstein - Barr virus chimeric antigen described in this application can be used for the prevention and / or treatment of EBV infection, and can also be used as a detection reagent for Epstein - Barr virus. When used for the prevention and / or treatment of EBV infection, the chimeric antigen can be used alone, can be used in combination with other antigens of Epstein - Barr virus, can be used in the form of a vaccine composition with different adjuvants, or can be used in the form of a multi - valent vaccine with different types of vaccine products.

[0008] The first object of the present invention is to provide a chimerically designed Epstein - Barr virus antigen, which is formed by connecting the three structural proteins gL, gH, and gp42 of Epstein - Barr virus in sequence using a linker to form an integral whole.

[0009] Furthermore, the gL structural protein is the soluble expression region in 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 as it.

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

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

[0012] Furthermore, the gH structural protein is the soluble expression region in 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 as it.

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

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

[0015] Furthermore, the gp42 structural protein is the soluble expression region in 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 as it.

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

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

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

[0019] In the present invention, a conventional linker structure can be selected for the linker.

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

[0021] Specifically, in certain embodiments, a GGGGSGGGG linker is used between gL and gH, and a GGSGG linker is used between gH and GP42.

[0022] Specifically, in certain embodiments, a cleavable GRSRR linker is used between gL and gH, and a GRSRRSRSGGS linker is used between gH and GP42.

[0023] In the present invention, when a cleavable linker is used, the prepared chimeric antigen can be normally expressed, and monomeric proteins can be obtained by enzymatic cleavage subsequently.

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

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

[0026] Furthermore, the disulfide bond mutations are disulfide bonds between the three structural proteins gL, gH, and gp42, or disulfide bonds within the structural proteins.

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

[0028] In the present 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 a cleavable linker. After the two aspects of modification, the antigen can still be normally expressed to prepare an antigen containing three components, indicating that the modification has a certain tolerance.

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

[0030] Specifically, in certain embodiments, the chimeric antigen can be used in combination with the gB protein of Epstein-Barr virus to increase immunogenicity.

[0031] The second object of the present invention is to provide a nucleic acid molecule encoding the Epstein-Barr virus chimeric antigen.

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

[0033] The third object of the present invention is to provide a vector, which contains the nucleic acid molecule.

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

[0035] The fifth object of the present invention is to provide a vaccine that contains the Epstein - Barr virus chimeric antigen.

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

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

[0038] The sixth object of the present invention is to provide a pharmaceutical composition that contains the Epstein - Barr virus chimeric antigen, the vaccine, the nucleic acid molecule, or the vector.

[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] The seventh object of the present invention is to provide the use of the Epstein - Barr 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 Epstein - Barr virus infection.

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

[0043] This application provides a chimerically designed Epstein - Barr virus antigen. Based on the technology of structural biology, a rational design of a multi - component vaccine is carried out to obtain a chimeric antigen that covers multiple component antigenic epitopes. By using a linker to connect the three proteins, gL, gH, and gp42 of the Epstein - Barr virus in sequence to form a whole, while maintaining the immunogenicity of the original multi - component antigen, the production and quality control processes are simplified, which helps to realize the industrialization of vaccine products. At the same time, the design of the multi - component chimeric vaccine in this application can stimulate a better neutralizing antibody titer compared with single - component vaccines, providing a new direction for clinical application and laying a foundation for the development of new products for preventing and blocking Epstein - Barr virus infection.

[0044] The above description is only an overview of the technical solutions and partial results of the present invention. In order to be able to more clearly understand the technical means of the present invention and implement it according to the content of the specification, the preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of different proteins after purification under reducing (R) and non-reducing (NR) conditions;

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

[0047] Figure 3 , Evaluation results of the immunogenicity (binding antibodies and neutralizing antibodies) of vaccine compositions prepared from different components. Detailed implementation mode

[0048] The present invention discloses a chimerically designed Epstein-Barr virus antigen and its application, and those skilled in the art can appropriately improve and implement it by referring to the content of this article. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0049] Terms:

[0050] In the present application, the term "Epstein-Barr virus" or "EBV" belongs to the subfamily Gammaherpesvirinae of the family Herpesviridae. The infection and latency of this virus are related to various lymphomas, epithelial cancers, and non-malignant diseases, such as various lymphoproliferative diseases, B / T / NK cell lymphomas, nasopharyngeal carcinoma (NPC), gastric cancer (GC), infectious mononucleosis (IM), oral hairy leukoplakia, systemic lupus erythematosus, and multiple sclerosis (MS).

[0051] In the present application, the term "chimeric antigen" generally refers to the tandem expression of multiple antigens through a linker. A sequence contains epitopes of multiple antigens, and a target protein presenting multiple antigen epitopes can be obtained through one recombinant expression. When used as a vaccine component, it can significantly increase immunogenicity and protectiveness. In the present application, the chimeric antigen refers to the design of connecting the Epstein-Barr virus gL, gH, and gp42 antigens through a linker.

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

[0053] In the present application, the term "codon optimization" generally means replacing one or more codons in the nucleic acid encoding a parental polypeptide with codons encoding the same amino acid residue but having different relative usage frequencies, so as to improve the expression of the nucleic acid encoding the polypeptide. In the present application, as long as the amino acid mutation is the same as that in the present application, all possible codons encoding the mutated amino acid are within the scope of protection of the present application.

[0054] In the present 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. For example, the transmembrane protein may include a secreted protein or a cell membrane protein. For example, the signal peptide may be synthesized in the form of a precursor polypeptide at the N-terminus of the transmembrane protein.

[0055] In the present application, the expression of a protein mutation site is generally expressed as "amino acid + amino acid position number + mutated amino acid". In the present application, the mutation may include, but is not limited to, addition, substitution, deletion, and / or removal of an amino acid. For example, the term "N84C" generally refers to the mutation of asparagine (N) at position 84 to cysteine (C).

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

[0057] In the application, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can be used to transform, transduce or transfect host cells so that the genetic elements carried by it can be expressed in the host cells. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal virus types used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). A vector may contain multiple elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain an origin of replication. The vector may also include components that assist its entry into cells or the integration of the target element into host cells, such as virus particles, liposomes, protein coats or integrases, but not limited to these substances.

[0058] In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or disorder. The pharmaceutical composition may comprise the Epstein-Barr virus chimeric antigen described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and / or the cell described in the present application, and optionally a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition may also comprise a suitable formulation 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 the present application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to the 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) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium, and / or non-ionic surfactants.

[0060] In the present application, the term "adjuvant" generally refers to any substance that aids 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 the present application, the adjuvant can be used to enhance the antigenicity of the Epstein - Barr virus (EBV) antigen. In certain embodiments, the adjuvant can comprise a suspension of minerals (such as alum, aluminum hydroxide, or phosphate). In certain embodiments, the adjuvant can include an oil - in - water emulsion. In certain embodiments, the adjuvant can include liposomes. In certain embodiments, the adjuvant can include immunostimulants such as MPL, CpG, Poly:IC, etc.

[0061] In the present application, the term "vaccine" refers to a pharmaceutical composition containing an immunogen capable of eliciting a prophylactic or therapeutic immune response in an individual. Generally, a vaccine elicits an antigen - specific immune response against a pathogen, such as a viral pathogen.

[0062] In the present application, the term "comprising" generally means including the expressly specified features, but not excluding other elements.

[0063] Based on structural biology and combined with immunological principles, the present application designs a novel protective vaccine with high safety, broad protection, and reliable preparation process. The EBV chimeric design antigen provided in the present application simultaneously and stably presents the antigenic epitopes of three proteins, which can increase the immunogenicity of the vaccine while simplifying the process of production scale - up and quality control. The EBV chimeric antigen described in the present invention can be used alone or in combination with other antigens, and has high immunogenicity when used as a vaccine or a vaccine component, can induce a relatively high level of neutralizing antibodies in immunized animals, can be used to prepare a vaccine for preventing or treating EBV infection, and can also be used as a reagent for detecting EBV.

[0064] Without being bound by any theory, the examples below are only for illustrating the EBV chimeric antigen, preparation method, quality characterization, and uses, etc. of the present application, and are not used to limit the scope of the invention of the present application.

[0065] The present invention is further illustrated below in conjunction with examples:

[0066] Example 1: Design of EBV protein mutants

[0067] The present invention relates to the design and evaluation of multiple EBV protein mutants. This example summarizes 5 typical representatives, including gp42 expressed alone, gH - gL expressed by chimeric expression of two components of gH and gL, gL - gH - gp42 expressed by chimeric expression of three components of gp42, gH, and gL, the three - component chimeric antigen gL - gH - gp42 - DS with disulfide bonds and cleavable linkers introduced, and the gB protein mutant expressed alone.

[0068] The separately expressed gp42 uses the publicly available sequence of GenBank: XOL08407.1 as a reference, and selects the amino acid sequence within the range of G62 - S251 of gp42 with the amino acid sequence shown in SEQ ID NO.3. A signal peptide sequence (MQLLCVFCLVLLWEVGA) derived from gH is added to the N-terminus, and a protein tag with six histidines is added to the C-terminus.

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

[0070] When designing the three-component chimeric antigen of gp42, gH, and gL, various different connection orders of the antigens were attempted, such as directly connecting gp42 to the C-terminus through a linker based on the gH-gL chimeric antigen, but a target protein that could be soluble expressed was not obtained. In this example, the preferred three-component chimeric antigen is based on a reasonable analysis of structural biology and uses the connection order of gL-gH-gp42. For the gL part, the amino acid sequence within the range of W24 - R135 in the SEQ ID NO.1 sequence is selected, for the gH part, the amino acid sequence within the range of A18 - A678 in the SEQ ID NO.2 sequence is selected, and for the gp42 part, the amino acid sequence within the range of R64 - S251 in the SEQ ID NO.3 sequence is selected. Fusion is carried out in the order of gL-gH-gp42 from the N-terminus to the C-terminus, where a GGGGSGGGG linker is used between gL-gH, and a GGSGG linker is used between gH-GP42.

[0071] Based on the design of the gL-gH-gp42 chimeric antigen, the gL-gH-gp42-DS mutant further investigated the effects on the chimeric antigen after introducing disulfide bonds and cleavable sites. For the gL part, the amino acid sequence within the range of W24 - H134 in SEQ ID NO.1 was selected; for the gH part, the amino acid sequence within the range of L20 - E675 in SEQ ID NO.2 was selected; for the gp42 part, the amino acid sequence within the range of K75 - S251 in SEQ ID NO.3 was selected. Fusion was carried out in the order of gL-gH-gpP42 from the N-terminus to the C-terminus. A cleavable GRSRR linker was used between gL and gH, and a GRSRRSRSGGS linker was used between gH and GP42. To further increase the stability of the gL and gH parts after cleavage, N84 in the gL sequence and G209 in the gH sequence were mutated to cysteine C.

[0072] The gB mutant used the sequence YP_401713.1 publicly available in the NCBI database as a reference, and selected the amino acid sequence within the range of M1 - D691. A GGSGGS linker and the Foldon trimerization domain of T4 phage containing a His tag (AIGGYIPEAPRDGQAYVRKDGEWVLLSTFLHHHHHH) were added to the C-terminus. The hydrophobic peptide segment at positions W193 - W196 in the fusion peptide was changed to a hydrophilic RVEA peptide segment to improve protein solubility. The potential furin cleavage site at positions R429 - R431 was mutated to GSG to avoid cleavage. The S467 site was mutated to proline P, and residues at positions R662 - Y664 were mutated to phenylalanine-cysteine-cysteine (FCC) to introduce disulfide bond formation between trimers to stabilize the trimeric structure of the gB protein.

[0073] The amino acid sequences of the above-mentioned protein mutants were entrusted to General Biotechnology Co., Ltd. for codon optimization using CHO as the host. A HindIII and kozark sequence (AAGCTTGCCGCCACC) was added to the 5' end of the gene, and a stop codon and NotI cleavage site (TGATAAGCGGCCGC) were added to the 3' end, followed by gene synthesis. The synthesized gene was cloned into the pKS001 mammalian cell vector (Zhongshan Kangsheng, A13201) containing a CMV promoter and SV40 polyA, and plasmid at the transfection level was extracted for subsequent cell transfection and protein expression testing.

[0074] Example 2: Expression of EBV protein mutants

[0075] Use HEK293F cells with rapid growth and good cell morphological state as the transfection host for small-volume transfection. Before transfection, adjust the viable cell density of the seeded cells to 6 million per milliliter, and use 20 mL of cell suspension for transfection for each project. For each project, use 20 μg of plasmid and 100 μg of PEI MAX transfection reagent (manufacturer: Polysciences, product number: 23966-1), dilute and mix them separately with 600 μL of OPM-293CD05 medium (manufacturer: OPM, product number: 81075-001), then slowly add the diluted transfection reagent to the diluted plasmid and gently mix. Incubate at room temperature for 8 minutes to prepare the plasmid complex. After incubation, slowly add the prepared plasmid complex to the cell suspension to be transfected, and place the cell suspension in a carbon dioxide shaking incubator at 37 °C and 8% CO2 for culturing at a rotation speed of 200 rpm. 18-24 hours after transfection, take samples to monitor the cell state and add 2 mL of BalanCD CHO Feed 4 (0.8X) (manufacturer: Fujifilm, product number: 94134) to each bottle of transfected cells. End the culture on the 4th day after transfection, and transfer the supernatant of the cell culture medium to the purification section for purification.

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

[0077] To obtain highly pure gB, gp42, gH-gL, gH-gL-gp42, and gH-gL-gp42-DS recombinant proteins respectively, the culture harvests of EBV proteins with different molecular designs expressed by HEK293F cells were isolated and purified. A two-step method was selected for isolation and purification, including a crude purification capture step for the culture harvest and a fine purification step by size exclusion chromatography.

[0078] For molecules purified crudely using affinity chromatography (such as gB, gp42, gH-gL-gp42), the designed sequences contain affinity tags. Dilute the culture harvest solution 1:1 with 1xPBS ph7.4, and use a Polar MC30-Ni Excel (Sepax 270630800) chromatography column pre-equilibrated with 1xPBS ph7.4 for capture. After capture, elute with 1xPBS ph7.4 containing 500 mmol / L imidazole to obtain the crudely purified protein; or use a HiTrap TM ProteinSelect TM (Cytiva17542152) chromatography column for capture. After capture, perform on-column retention, and elute with 1xPBS ph7.4 to obtain the crudely purified protein.

[0079] For the crude purification of molecules (such as gH-gL, gH-gL-gp42-DS) using cation exchange chromatography, without any tags in the designed sequence, after adjusting the pH with dilute hydrochloric acid and the conductivity with 20 mmol / L phosphate buffer, capture was performed using a CEX (UniGel 80SP) (Nano Micro 04082-080100-2050) chromatography column pre-equilibrated with 20 mmol / L phosphate buffer. After capture, elution was carried out using 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, and HiLoad 16 / 600 Superdex200pg (Cytiva 28989335) or Superdex 200 Increase 10 / 300GL (Cytiva 28990944) was selected according to the content. Before loading, the chromatography column system was equilibrated to a stable baseline with 1xPBS pH7.4, and then the concentrated sample of the crudely purified protein was added using a syringe. After the products with different retention times flowed out, the target protein fractions were collected to obtain highly pure EBV recombinant protein.

[0081] As Figure 1 shown, highly pure target proteins were obtained after the separation and purification of EBV recombinant proteins with different molecular designs.

[0082] Example 4: Analysis of the particle size distribution of the designed chimeric EB antigen

[0083] To analyze the size distribution of the designed EB chimeric antigen, this experiment used SEC-HPLC method to analyze the purified sample. The specific implementation method was as follows: The chromatography column was a NanoChrom BioCore SEC-300 (5μm, 7.8*300mm) chromatography column, the mobile phase was 20 mM PB + 300 mM NaCl pH7.4, the flow rate was 0.5 ml / min, the detection wavelength was 280 nm, and isocratic elution was performed. Separation and detection were carried out through the Shimadzu LC-20A system, and the proteins were eluted and separated according to the molecular size of the components to be measured.

[0084] As Figure 2 shown, the designed chimeric antigen had a single peak position and a symmetric peak shape, presenting a good homogeneous conformation.

[0085] Example 5: Immune response of the chimerically designed EB antigen in mice

[0086] To compare the immunogenicity of different protein components of EBV immunized alone or in combination, female BALB / c mice at 5-6 weeks of age were randomly grouped, with 6 mice in each group, for a total of 7 groups, including the gB, gp42, gH-gL, gL-gH-gp42, gH-gL+gp42, gB+gL-gH-gp42 antigen groups and the normal saline control group. Immunization was carried out by intramuscular injection at 0 and 21 days, 50 μl per mouse, and sera were collected at 42 days for detection of binding antibody and neutralizing antibody titers.

[0087] Table 1 Prescription Information

[0088]

[0089]

[0090] The specific preparation method of the vaccine composition is as follows: First, CpG7909 (Guangzhou Ribobio Co., Ltd.) is adsorbed onto aluminum hydroxide adjuvant (CRODA, product number AJV3012) (here the content of aluminum hydroxide adjuvant is essentially the content of aluminum element), and then the antigen is added to the CpG7909 / aluminum hydroxide sample and adsorbed at room temperature for 30 minutes before use. If not used immediately, it is stored at 2-8 °C.

[0091] The method for detecting the binding antibody titer is as follows: The indirect ELISA method is used for detecting the binding antibody titer. The antigen is diluted to 2 μg / ml with buffer and coated on a 96-well plate at 100 μl per well, and incubated overnight at 2-8 °C; after washing with PBST, blocking solution (PBST containing 5% skim milk powder) is added at 300 μl per well and incubated at 25 °C for 1 h; after washing with PBST, serum test samples (after being diluted to a certain multiple with the blocking solution and then serially diluted 2-fold, for a total of 11 dilution degrees, with duplicate wells set, and the starting dilution multiple of the normal saline group is 100) and blank controls (blocking solution, 4 wells are set per plate) are added at 100 μl per well and incubated at 25 °C for 2 h; after washing, HRP-labeled secondary antibody (BETHYL, A90-116P, diluted 5000-fold with the blocking solution) is incubated at 100 μl per well and incubated at 25 °C for 1 h; finally, TMB chromogenic solution is added at 100 μl per well and incubated at 25 °C for 10 min. After the reaction is terminated, the absorbance value at 450 nm is read in an enzyme-linked immunosorbent assay reader. Data screening and analysis are carried out according to 2.1 times the average absorbance value greater than the blank control in the same-day experiment as the limit value (cut-off value) of this experiment, and data statistics are performed using GraphPad Prism 8.0 software.

[0092] The detection method of neutralizing antibody titer is as follows: (1) Serum serial dilution: In a V-bottom 96-well plate, the first well is diluted 1:10 (diluted with serum-free RPMI1640, and the actual dilution factor of the first well after adding the virus is 1:20), and serum-free RPMI1640 is serially diluted 8 gradients by three times (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°C for 2 hours. Positive control: 20 μl of serum-free RPMI1640 (without adding serum) + 20 μl of virus. (3) Akata-neg plating: Plate Akata-neg cells into a 96-well plate at a density of 10,000 cells / well / 160 μl; (4) Add the 40 μl virus + serum mixture incubated for 2 hours above to the cell culture wells (a total of 200 μl) and culture for 48 hours. (5) Separate the cells in each well into an EP tube and detect the GFP positive rate by flow cytometry. (6) Fitting: Calculate the ID50 by four-parameter non-linear fitting with GraphPad Prism.

[0093] As Figure 3 shown, after two vaccinations, compared with the normal saline group, the binding antibody levels of each antigen group were significantly increased, the differences between vaccine groups were small, but the differences in neutralizing antibody titer levels between groups were significant. After the second vaccination, the neutralizing antibody titers of the multi-component antigen groups were higher than those of the single-component antigen groups. Among the multi-component antigen groups, the two groups containing the gL-gH-gp42 three-component antigen had the highest neutralizing antibody titers. Among them, the neutralizing antibody titer level of the gL-gH-gp42 chimeric antigen of the present application was comparable to that of the antigens expressed separately and then used in combination.

[0094] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. It should be pointed out that for those of ordinary skill in the art of this technology, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A chimeric Epstein-Barr virus antigen, characterized in that, The chimeric antigen is formed by connecting three structural proteins, namely gL, gH, and gp42 of Epstein-Barr virus, in sequence using a linker to form an integral whole.

2. The EB virus antigen according to claim 1, wherein The gL structural protein is the soluble expression region of the gL protein with the amino acid sequence as shown in SEQ ID NO.1, or a protein with more than 95% homology and the same or substantially the same immunogenicity as it.

3. The Epstein-Barr virus antigen according to claim 1, wherein The gH structural protein is the soluble expression region of the gH protein with the amino acid sequence as shown in SEQ ID NO.2, or a protein with more than 95% homology and the same or substantially the same immunogenicity as it.

4. The EB virus antigen according to claim 1, wherein, The gp42 structural protein is the soluble expression region of the gp42 protein with the amino acid sequence as shown in SEQ ID NO.3, or a protein with more than 95% homology and the same or substantially the same immunogenicity as it.

5. The Epstein - Barr virus antigen according to any one of claims 1 to 4, characterized in that, The linker is a fragment of 1 to 30 amino acids.

6. The EB virus antigen according to claim 5, wherein The structure of the linker is (GGS)n, where n is an integer between 1 and 10.

7. The Epstein-Barr virus antigen according to any one of claims 1 to 4, characterized in that, The chimeric antigen may further include at least one disulfide bond mutation.

8. The EB virus antigen according to claim 7, wherein The disulfide bond mutation is to mutate N84 in the gL sequence and G209 in the gH sequence into cysteine C.

9. A nucleic acid molecule encoding the Epstein-Barr virus chimeric antigen according to any one of claims 1 to 8.

10. A carrier, characterized in that, The vector contains the nucleic acid molecule according to claim 9.

11. A cell, characterized in that, The cell expresses the Epstein-Barr virus chimeric antigen according to any one of claims 1 to 8, or contains the nucleic acid molecule according to claim 9, or contains the vector according to claim 10.

12. A vaccine, characterized in that, The vaccine contains the Epstein-Barr virus chimeric antigen according to any one of claims 1 to 8.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the Epstein-Barr virus chimeric antigen according to any one of claims 1 to 8, the vaccine according to claim 12, the nucleic acid molecule according to claim 9, or the vector according to claim 10.

14. Use of the Epstein-Barr virus chimeric antigen according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, the vector according to claim 10, the cell according to claim 11, the vaccine according to claim 12, or the pharmaceutical composition according to claim 13 in the preparation of products for detecting, preventing, and / or treating Epstein-Barr virus infection.

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