CN-121975021-A - Fusion immunogen and application thereof in preparation of EBV infected self-replicating mRNA vaccine

Abstract

The invention relates to a fusion immunogen and application thereof in preparing an EBV infected self-replicating mRNA vaccine, belonging to the technical field of biological medicine. The fusion immunogen consists of a signal peptide, an EB virus latency protein, a connector and MITD structures. The EB virus latency protein is selected from one or more of EBNA1, EBNA3A, LMP1 and LMP 2. The fusion immunogen can be used for constructing a self-replicating mRNA vaccine, can induce organisms to generate specific humoral immunity and cellular immunity reaction, and is suitable for preventing and treating EBV related diseases.

Inventors

• XIONG CHANGYUN
• Xu Zhedi
• WANG YOURU
• YANG YONGJUN
• CHEN GENG
• ZHOU RUI
• SONG WEN
• WANG XUELIAN
• WANG YIXIN
• MA LI

Assignees

• 宁波君健生物科技有限公司

Dates

Publication Date

20260505

Application Date

20260130

Claims (10)

1. 1. A fusion immunogen is characterized by comprising a signal peptide, an EB virus latency protein, a connector and MITD, wherein the EB virus latency protein is one or more of EBNA1, EBNA3A, LMP1 and LMP2, and the EB virus latency protein has an amino acid sequence shown in any one of SEQ ID NO 1-4.
2. 2. The fusion immunogen of claim 1, wherein EBNA1 has an amino acid sequence as shown in SEQ ID NO. 1, EBNA3A has an amino acid sequence as shown in SEQ ID NO. 2, LMP1 has an amino acid sequence as shown in SEQ ID NO. 3, and LMP2 has an amino acid sequence as shown in SEQ ID NO. 4.
3. 3. The fusion immunogen of claim 1, wherein the signal peptide comprises any one or more of tPA signal peptide, MIgG2a signal peptide, nuclear localization signal peptide, alpha-factor signal peptide, ig kappa light chain signal peptide, and Secrecon signal peptide.
4. 4. The fusion immunogen of claim 1, wherein the linker has the amino acid sequence shown in SEQ ID NO. 6.
5. 5. The fusion immunogen of claim 1, wherein the amino acid sequence of MITD is set forth in SEQ ID NO. 7.
6. 6. The fusion immunogen according to claim 1, wherein the fusion immunogen has an amino acid sequence shown in any one of SEQ ID NOs 8 to 10.
7. 7. A nucleic acid molecule encoding the fusion immunogen of any one of claims 1-6.
8. 8. Use of the fusion immunogen of any one of claims 1-6 or the nucleic acid molecule of claim 7 in the manufacture of a medicament for the prevention or treatment of epstein barr virus infection.
9. 9. A self-replicating mRNA vaccine for epstein barr virus, comprising the fusion immunogen of any one of claims 1-6 or the nucleic acid molecule of claim 7.
10. 10. The method for preparing the self-replicating mRNA vaccine according to claim 9, wherein the method for preparing the mRNA vaccine comprises the steps of: s1, cloning nucleic acid molecules encoding fusion immunogens to a self-replicating vector, constructing a self-replicating mRNA plasmid, and introducing the self-replicating mRNA plasmid into a host cell to obtain a genetically engineered cell; s2, culturing the genetically engineered cells, extracting from replicative mRNA plasmids, and performing enzyme digestion to obtain linearized plasmids; S3, synthesizing a self-replicating mRNA molecule in vitro by taking the linearization plasmid as a template, and purifying to obtain a self-replicating mRNA stock solution; s4, preparing a lipid mixture containing cationic lipid and auxiliary lipid, dissolving in a solvent and filtering to form an organic phase, dissolving the self-replicating mRNA stock solution in a buffer medium to form a water phase, mixing the organic phase with the water phase to form an mRNA-LNP initial preparation, and concentrating and changing the solution to obtain the self-replicating mRNA vaccine.

Description

Fusion immunogen and application thereof in preparation of EBV infected self-replicating mRNA vaccine Technical Field The invention belongs to the technical field of biological medicine, and particularly relates to a fusion immunogen and application thereof in preparation of an EBV (infectious bovine serum albumin) infected self-replicating mRNA vaccine. Background Epstein-Barr virus (EBV) is related to 2% of human malignant tumors such as nasopharyngeal carcinoma, gastric cancer, various lymphomas and the like, and can also cause diseases such as infectious mononucleosis, chronic active EBV infection and the like, and no specific treatment method aiming at the EBV exists at present. The existing treatment scheme mainly focuses on controlling symptoms and supporting treatment, and for EBV related tumors, chemotherapy and radiotherapy are main treatment means, but have the problems of large side effect, easy recurrence and the like. EBNA1 and EBNA3A are nuclear antigens encoded by the epstein barr virus, in which the EBV proteins expressed by EBNA1 in all EBV-associated tumors play an important role in the replication and maintenance of the epstein barr virus genome in host cells. Due to the broad expression of EBNA1 in EB virus-related tumors, it is a potential target antigen in immunotherapy against EBV-related malignancies. However, the natural immunogenicity of EBNA1 is weak, and how to enhance the immunogenicity of EBNA1 is a key difficulty in developing a vaccine using EBNA1 or EBNA3A as a core antigen. The mRNA vaccine has the advantages of high safety, short research and development period, simple and convenient production, strong immune response induction and the like, and the self-replicating mRNA can be self-replicated in cells for a long time, so that the expression quantity of target protein is increased, the drug effect is improved, the acting time is prolonged, and the mRNA vaccine is a high-quality immunotherapy carrier. Based on this, the potential for developing EBV therapeutic mRNA vaccines is enormous, but challenges in antigen selection, delivery systems, overcoming immune escape, etc. are faced. Self-replicating mRNA is a new iteration of the mRNA technology in the field of mRNA technology, and has the ability to self-replicate for a long period of time in recipient cells, which can produce more mRNA molecules, thereby increasing the expression level of the target protein. Based on the mechanism, mRNA is self-replicated to become a most potential drug carrier for antigen-specific immunotherapy, which can improve the efficacy of vaccines or drugs, prolong the time of the drugs acting in vivo, and show the effects of low injection frequency and low injection quantity and long-acting therapy. The related patent documents: Foreign china, publication No. CN119604618a, publication date 2025.03.11, which discloses vaccines based on viral vectors for delivering antigens against infectious diseases. In particular, it relates to a recombinant modified vaccinia virus ankara (MVA) encoding EBV antigens that cause Infectious Mononucleosis (IM) and different cancer types. The invention also relates to the medical use of said recombinant MVA for the prevention of diseases caused by EBV. Foreign China is published under publication No. CN118308379A, publication date 2024.07.09, which discloses an Epstein-Barr virus antigen construct. The invention provides epstein-barr virus antigen polynucleotides, polypeptides and vectors, and immunogenic compositions comprising the same. It involves the use of epstein-barr virus antigen constructs to generate vaccines for the treatment and prevention of epstein-barr virus infections and epstein-barr virus related diseases such as multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. The prior art represented by the foregoing documents has at least the following technical problems or drawbacks that have not been solved: The related evidence is that the patent document with the publication number of CN119604618A does not contain core tumor antigens in the EBV latent infection stage such as EBNA1, LMP2 and the like, envelope proteins in the cracking stage such as gp350, gH, gL and the like are difficult to target tumor cells in latent infection, the immune treatment correlation is insufficient, multiple antigens are simply co-expressed, the cooperative design such as fusion expression and the like is not adopted, a single antigen immunogenic short plate cannot be broken through the functional complementation among the antigens, and the whole immune activation effect is difficult to meet the treatment requirement of the EBV related tumor. The invention selects three sequences of EBNA3A, EBNA A-EBNA1-LMP2-LMP1 and EBNA3A-EBNA1-LMP 2. Applicants have designed self-replicating mRNA molecules for the treatment of EBV tumors using self-replicating mRNA techniques that express EBNA3A, EBNA A-EBNA1-LMP2-LMP1 and EBNA3A-EBNA1-LMP2 fusion proteins. The inven