Mycobacterium tuberculosis mRNA vaccine as well as construction method and application thereof

Abstract

The invention discloses a mycobacterium tuberculosis mRNA (messenger Ribonucleic Acid) vaccine as well as a construction method and application thereof, and belongs to the technical field of biological medicines. The technical problem to be solved is to provide a novel mycobacterium tuberculosis mRNA vaccine which is low in dosage, high in safety and lasting in immune memory. According to the technical scheme, the preparation method is characterized by comprising the following steps: carrying out tandem expression on antigens for coding Hsp65 and Mpt83, constructing mRNA molecules by combining tPA signal peptide (SEQ ID NO: 1), an MITD sequence (SEQ ID NO: 5) and flexible Linker (SEQ ID NO: 6-7), and carrying out codon optimization (an mRNA secondary structure and GC content) and a lipid nanoparticle (LNP) encapsulation process to finally obtain the novel mycobacterium tuberculosis mRNA vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a Mycobacterium tuberculosis mRNA vaccine, its construction method, and its application. Background Technology

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). The incidence and mortality rates of TB remain consistently high. Therefore, there is an urgent need to develop new and more effective methods for TB prevention.

[0003] Currently, Bacille Calmette-Guérin (BCG) is the only widely used tuberculosis vaccine. While BCG provides some protection in newborns, its protective effect diminishes significantly in adolescents and adults, and its protective efficacy against extrapulmonary tuberculosis is limited. Furthermore, BCG has limitations, such as being unsuitable for immunocompromised individuals and the potential for cross-reactivity with tuberculosis diagnosis. Therefore, BCG does not fully meet the needs for tuberculosis prevention.

[0004] To overcome the limitations of existing vaccines, researchers have been exploring the development of novel tuberculosis vaccines. These vaccines aim to provide broader protection, including against pulmonary tuberculosis in adults, and to improve vaccine safety and efficacy.

[0005] Relevant patent documents retrieved: The publication, published in China (CN112062859A) on December 11, 2020, discloses a chimeric antigen receptor for pathogen clearance and its applications, which comprises: (a) an extracellular domain including one or more antigen-binding regions specific to pathogen-associated antigens; (b) a transmembrane domain; and (c) a "eat" (likely referring to a specific antigen-binding domain). At least one copy of the intracellular signaling domain of the "me" signaling receptor can specifically bind to pathogen antigens and activate endogenous silent phagocytic clearance signals. When the pathogen is Mycobacterium tuberculosis, especially Mycobacterium tuberculosis infection: secretory antigen SssA; molecular chaperone DnaK, cell surface lipoprotein Mpt83, lipoprotein P23, phosphate transport system permease protein pstA, 14kDa antigen, fibronectin-binding protein CFbpC1, alanine dehydrogenase TB43, glutamine synthase 1, ESX-1 protein, protein CFP10, TB10.4 protein, protein MPT83, protein MTB12, protein MTB8, Rpf-like protein, protein MTB32, protein MTB39, crystal protein, heat shock protein HSP65, and protein PST-S can serve as antigens.

[0006] The retrieved relevant non-patent literature was compared with publicly reported immunological results from several major TB vaccine candidates (e.g., M72 / AS01E, H4:IC31, H56:IC31, and the MVA85A series). These studies typically use GMC / seroconversion of anti-antigen IgG and PBMC ICS (CD4+) as indicators. + Phenotype / IFN-γ) and IFN-γELISPOT (SFU / 10) 6 Using PBMC (or pg / ml after in vitro stimulation) as an immunogenicity indicator, the VEEV vector mRNA vaccine described in this invention, under the experimental methods and detection system used in this study, showed a binding antibody endpoint titer as high as 1:1,000,000, accompanied by significant T cell activation (FACS / ICS) and a high level of IFN-γ ELISPOT response. It should be noted that the compared publicly available data differ in methodological parameters such as antigen selection, ELISA endpoint definition, sample source, adjuvant, and time points; therefore, the literature listed below is for reference only and not a verbatim equivalent comparison.

[0007] literature: 1. Final Analysis of a Trial of M72 / AS01EVaccine to PreventTuberculosis, DOI:10.1056 / NEJMoa1909953 2. Prevention of M. tuberculosis Infection with H4:IC31 Vaccine or BCGRevaccination, DOI: 10.1056 / NEJMoa1714021 3. Protection and Polyfunctional T Cells Induced by Ag85B-TB10.4 / IC31® against Mycobacterium tuberculosis Is Highly Dependent on the AntigenDose, https: / / doi.org / 10.1371 / journal.pone.0005930 4. Safety, immunogenicity, and efficacy of the candidate tuberculosisvaccine MVA85A in healthy adults infected with HIV-1: a randomized, placebo-controlled, phase 2 trial, DOI: 10.1016 / S2213-2600(15)00037-5 5. Testing the H56 Vaccine Delivered in 4 Different Adjuvants as aBCG-Booster in a Non-Human Primate Model of Tuberculosis, DOI: 10.1371 / journal.pone.0161217 Journal or book title: *FEMS Immunology and Medical Microbiology*; Article title: "A novel vaccine strategy to induce mycobacterial antigen-specific Th1 responses by utilizing the C-terminal domain of heat shock protein 70."; Volume: 61, 2 (2011). This paper, 189-96, published on January 18, 2011, discloses a new direction for MPT51-HSP70C fusion vaccines as candidates for tuberculosis vaccines. It describes fusing MPT51 with the full-length (HSP70F), N-terminus (HSP70N), or C-terminus (HSP70C) of HSP70 to construct plasmid DNA vaccines. By detecting IFN-γ (Th1 cytokine) levels using ELISA and IFN-γ and iNOS expression using RT-PCR, it was found that the C-terminal domain of HSP70 plays a crucial role in enhancing antigen-specific Th1 responses. Utilizing the C-terminal domain of Mycobacterium tuberculosis HSP70 enhances antigen-specific Th1 immune responses, thereby improving the protective efficacy of the vaccine against tuberculosis.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: CD8 cells could not be activated. The relevant evidence is found in the first paragraph of the non-patent literature results section: "CD4 T cells from C57BL / 6 mice immunized with HSP70F-MPT51 produced a significantly higher amount of IFN-γ than those immunized with MPT51... The addition of HSP70F to MPT51 did not show a significant enhancing effect on the antigen-specific CD8 response, although CD8 T cells from BALB / c mice immunized with HSP70F-MPT51 showed a slightly higher IFN-γ production than those immunized with MPT51." In other words, while the vaccine successfully increased IFN-γ secretion from CD8 T cells, it did not significantly activate antigen-specific CD8 cells. + T cell response.

[0009] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: (1) Hsp65 and Mpt83 were screened as antigens from a large number of Mycobacterium tuberculosis antigens commonly used in existing technologies. They significantly activated specific T cell immune responses. The screening range was wide and the experimental results did not meet the linear conditions.

[0010] (2) The amino acid sequences of the tPA signal peptide and MITD sequence were optimized based on the selected antigens, and codon optimization was performed based on the optimization results. During optimization, it is necessary to consider not only the protein structure, but also whether the secondary structure of its mRNA will be affected. There are many screening indicators. Summary of the Invention

[0011] The purpose of this invention is to provide: A Mycobacterium tuberculosis mRNA vaccine, its construction method and application, and related technologies, to solve technical problems such as improving the activation of specific T cell immune responses, inducing efficient humoral and cellular immunity, enhancing the broad spectrum of immunity, and improving antigen expression and presentation efficiency, or combinations thereof.

[0012] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0013] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0014] The definition of standard chemical terms can be found in the reference "Experimental Guide to Antibody Preparation and Use: Science Press: 2010:1".

[0015] Unless otherwise specified, conventional methods within the scope of the art, such as strain transformation experiments, protein concentration determination, sequencing verification, and establishment of standard curves, shall be used.

[0016] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0017] The terms "optional / arbitrary" or "optionally / arbitrarily" mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation. For example, according to the definition below: the excipients of a vaccine include any one or more of the following: adjuvant, stabilizer, preservative, buffer. This means that the excipients in the vaccine include adjuvants; or the excipients in the vaccine include stabilizers; or the excipients in the vaccine include preservatives; or the excipients in the vaccine include adjuvants, stabilizers, and preservatives.

[0018] The term "tPA signal peptide" used in this article refers to the N-terminal secretory signal peptide of human tissue plasminogen activator (tPA), which mediates the entry of nascent polypeptide chains into the endoplasmic reticulum-Golgi apparatus secretion pathway, thereby enabling protein transmembrane transport.

[0019] The term “MITD sequence” used in this article refers to the abbreviation of the MHC-I trafficking domain, which is a sequence derived from the transmembrane-cytoplasmic domain of the HLA-DM molecule. It can promote the binding of antigens to MHC-I molecules in the endoplasmic reticulum and enhance their presentation to the cell surface.

[0020] The term "self-replicating vector" as used in this article refers to a vector that contains a non-structural protein gene, a subgenomic promoter, and a foreign gene insertion site, and can amplify the target transcript intracellularly via RNA-dependent RNA polymerase.

[0021] The term "codon optimization" used in this article refers to replacing rare codons with synonymous codons that are abundant in host cell tRNAs in gene coding regions, and adjusting GC content and mRNA secondary structure to improve transcript stability and translation efficiency.

[0022] In a first aspect, the present invention provides: a fusion protein.

[0023] It includes the following technical features: Hsp65 antigen, Mpt83 antigen, binding tPA signal peptide, and MITD sequence.

[0024] Among them, the technical feature Hsp65 is selected from sequences that have more than 85% homology with SEQ ID NO.4.

[0025] The preferred technical feature Hsp65 is a sequence with more than 90% homology to SEQ ID NO.4.

[0026] The technical feature Hsp65 is further preferably a sequence with more than 98% homology to SEQ ID NO.4.

[0027] Among them, the technical feature Hsp65 is further preferably: SEQ ID NO.4.

[0028] Among them, the technical feature Mpt83 is selected from sequences that have more than 85% homology with SEQ ID NO.3.

[0029] The preferred technical feature Mpt83 is a sequence with more than 90% homology to SEQ ID NO.3.

[0030] The technical feature Mpt83 is further preferably a sequence with more than 98% homology to SEQ ID NO.3.

[0031] Among them, the technical feature Mpt83 is further preferably: SEQ ID NO.3.

[0032] The technical feature tPA signal peptide is selected from sequences that have more than 95% homology with SEQ ID NO.1.

[0033] The preferred technical feature of the tPA signal peptide is a sequence with more than 96% homology to SEQ ID NO.1.

[0034] The tPA signal peptide is further preferably a sequence with more than 98% homology to SEQ ID NO.1.

[0035] The technical feature tPA signal peptide is further preferably: SEQ ID NO.1.

[0036] The MITD sequence, a technical feature, is selected from sequences that share more than 95% homology with SEQ ID NO.5.

[0037] The preferred technical feature MITD sequence is a sequence with more than 96% homology to SEQ ID NO.5.

[0038] The MITD sequence is further preferably a sequence with more than 98% homology to SEQ ID NO.5.

[0039] The preferred technical feature MITD sequence is SEQ ID NO.5.

[0040] The technical feature fusion protein sequence is selected from sequences with more than 85% homology to SEQ ID NO.9.

[0041] The preferred technical feature fusion protein sequence is one that has more than 90% homology with SEQ ID NO.9.

[0042] The preferred embodiment of the technical feature fusion protein sequence is a sequence with more than 95% homology to SEQ ID NO.9.

[0043] The preferred fusion protein sequence is SEQ ID NO.9.

[0044] Among them, the technical features of Hsp65 antigen, Mpt83 antigen, binding tPA signal peptide and MITD sequence are linked by direct linking or linking through a linker.

[0045] Among them, the technical features of the linker include, but are not limited to, the flexible linker.

[0046] Among them, the technical features of the flexible linker include, but are not limited to: (G) x S y ) nx≤4, y≤2, n≤6.

[0047] Among them, the Mpt83 antigen and Hsp65 antigen are preferably connected by a flexible Linker.

[0048] The flexible linker sequence is preferably SEQ ID NO:6: GGSGGGSGGGSGGGS.

[0049] Preferably, a flexible linker is used to connect the Mpt83 antigen or Hsp65 antigen to the MITD sequence.

[0050] The flexible linker sequence is preferably SEQ ID NO:7: GGSGGGSGGGS Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred approach: tandem expression of Hsp65 and Mpt83 antigens combined with the tPA signal peptide. This approach, while addressing the technical issue of "increasing antibody titer," further addresses the technical issue of "enhancing protein secretion efficiency."

[0051] The second preferred approach involves the tandem expression of Hsp65 and Mpt83 antigens combined with an optimized tPA signal peptide. This approach, building upon the existing technical problem of "enhancing protein secretion efficiency," further addresses the issue of "enhancing protein secretion efficiency."

[0052] The third preferred approach involves the tandem expression of Hsp65 and Mpt83 antigens combined with an optimized tPA signal peptide and MITD sequence. This approach, while addressing the technical issue of "enhancing protein secretion efficiency," further addresses the technical issue of "improving the strength and quality of cellular immune responses."

[0053] Secondly, the present invention provides a nucleic acid encoding the above-mentioned fusion protein.

[0054] The technically characteristic nucleic acid sequence is selected from sequences encoding any of the above-mentioned fusion proteins, including DNA and RNA.

[0055] The DNA sequence of the technically characteristic nucleic acid is selected from a sequence that has more than 50% homology with SEQ ID NO.10.

[0056] The preferred technical feature DNA sequence is one that has more than 85% homology with SEQ ID NO.10.

[0057] The technical characteristic DNA sequence is further preferably a sequence with more than 95% homology to SEQ ID NO.10.

[0058] The preferred technical feature DNA sequence is SEQ ID NO.10.

[0059] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the second aspect of the present invention includes: First preferred option: DNA sequence as shown in SEQ ID NO.10.

[0060] This technical solution not only solved the technical problem of "enhancing the strength and quality of cellular immune responses", but also further solved the technical problem of "increasing gene expression levels".

[0061] Thirdly, the present invention provides an expression carrier.

[0062] The expression vector expresses any of the above-mentioned fusion proteins or carries any of the above-mentioned nucleic acids.

[0063] The technical features include at least one of the following elements: viral replication enzyme, eukaryotic promoter, stop codon, and self-replicating vector.

[0064] Among them, the technical characteristics of viral replicase include, but are not limited to: HIV-1 reverse transcriptase, Ad DNA polymerase, HSV DNA polymerase, Vaccinia DNA polymerase, flavivirus NS5, and small RNA virus 3D polymerase.

[0065] The preferred technical feature of the viral replication enzyme is either flavivirus NS5 or small RNA virus 3D polymerase.

[0066] The viral replication enzyme, a key technical feature, is further preferably a small RNA virus 3D polymerase.

[0067] Among them, the technical feature of viral replicase is further preferably VEEV (Venezuelan equine encephalitis virus) replicase.

[0068] Among them, the technical characteristics of eukaryotic promoters include, but are not limited to: CMV, EF1α, CAG, SV40, Alb (liver), GFAP (astrocytes), Tet-On / Tet-Off, UAS / Gal4, RSV LTR, and HTLV LTR.

[0069] Among them, the preferred eukaryotic promoters are: CMV promoter, EF1α promoter, and CAG promoter.

[0070] Among them, the eukaryotic promoter with the technical feature is further preferably the CMV promoter.

[0071] Among them, the eukaryotic promoter with the technical feature is further preferably: the enhanced CMV promoter (eCMV promoter).

[0072] Among them, the technical feature stop codons include, but are not limited to: UAA, UAG, UGA, and UAGA.

[0073] The preferred stop codon for the technical feature is either UAA or UGA.

[0074] The technical feature termination codon is further preferably UAA.

[0075] Among them, the technical feature termination codon is further preferably: a double UAA termination codon (to increase translation termination efficiency).

[0076] Among them, the technical features of self-replicating vectors include, but are not limited to: Sindbis / SFV replicon, EBVoriP episome, mammalian artificial chromosome (MAC), piggyBac transposon, and AAV self-replicating particles.

[0077] The preferred technical feature of the self-replicating vector is an alphavirus replicon (such as SFV or Sindbis).

[0078] The self-replicating vector of the technical feature is further preferably an SFV replier.

[0079] The technical feature of the self-replicating vector is further preferably the VEEV replicon.

[0080] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the third aspect of the present invention includes: The first preferred option: the self-replicating vector is JJ-SaRNA-2. This invention provides a self-replicating RNA molecule based on the Venezuelanequine encephalitis virus (VEEV) replicon backbone and its applications. The RNA molecule comprises: a 5' untranslated region; sequences encoding VEEV unstructural replication proteins nsP1, nsP2, nsP3, nsP4 (or their functionally equivalent variants); a 26S subgene promoter downstream of the replication protein coding region; and a heterologous antigen coding sequence linked downstream of the subgene promoter. The RNA may further comprise a 3' untranslated region and a poly(A) tail or other equivalent stabilizing elements. To reduce the potential cytotoxicity of the replicon to host cells, the replication protein may contain known attenuated or functionally preserved mutations or variants. The RNA may be delivered as naked RNA or in a vector system (e.g., lipid nanoparticles); it may also be delivered in vitro or in vivo as replicon particles (VRPs) by providing structural proteins in packaging cells. The present invention also covers DNA templates encoding the replicon RNA, their preparation, and methods for inducing immune responses in mammals.

[0081] This technical solution not only solved the technical problem of "improving gene expression levels", but also further solved the technical problem of "stimulating a stronger immune response with lower doses and for a longer duration".

[0082] Fourthly, the present invention provides: an LNP encapsulation method for any of the above-mentioned fusion proteins and / or nucleic acids.

[0083] This includes technical features such as the materials used for encapsulation, the proportions of the materials, the pH of the encapsulation material, and the ratio of the aqueous phase to the organic phase.

[0084] The materials used for encapsulating the technical features are selected from: Dlin-MC3-DMA, SM-102, ALC-0315, 306Oi10, 503Oi10, distearate phosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), cholesterol, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), PEG2000-PE, and C14-PEG.

[0085] The preferred materials used for encapsulating the technical features are: Dlin-MC3-DMA, SM-102, ALC-0315, 306Oi10, 503Oi10, DSPC, DOPE, cholesterol, and DMG-PEG2000.

[0086] The materials used for encapsulating the technical features are further preferably: Dlin-MC3-DMA, SM-102, ALC-0315, 306Oi10, 503Oi10, DSPC, cholesterol, and DMG-PEG2000.

[0087] The materials used for encapsulating the technical features are further preferably: Dlin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000.

[0088] The proportions of the technical characteristic materials used (Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000) are selected from: 40-60: 5-10: 30-50: 1-10.

[0089] The preferred ratio of the technical feature materials used is 45-55:5-9:35-45:2-5.

[0090] The preferred ratio of the technical feature materials used is 49-51:6-8:39-41:2-4.

[0091] Fifthly, the present invention provides lipid nanoparticles prepared by the above-described lipid nanoparticle encapsulation method.

[0092] In a sixth aspect, the present invention provides the use of any of the above-mentioned fusion proteins, nucleic acids, expression vectors or lipid nanoparticles in the preparation of medicaments for the prevention and / or treatment of Mycobacterium tuberculosis-related diseases.

[0093] In a seventh aspect, the present invention provides: a vaccine comprising any of the above-described fusion proteins, nucleic acids, expression vectors, or lipid nanoparticles.

[0094] This includes technical features such as auxiliary materials and application methods.

[0095] The technical feature excipients are selected from: adjuvants, stabilizers, preservatives, and buffer solutions.

[0096] The application methods for the technical features are selected from: intramuscular injection (deltoid or vastus lateralis), subcutaneous injection, oral administration, and nasal spray.

[0097] The preferred methods of application of the technical features are: intramuscular injection, subcutaneous injection, and nasal spray.

[0098] The preferred method of application of the technical feature is intramuscular injection or subcutaneous injection.

[0099] The preferred method of application of the technical feature is intramuscular injection.

[0100] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first priority option: The vaccine also includes excipients. This technical solution, based on solving the technical problems of "high encapsulation rate and good stability", further solves the technical problems of "stabilizing vaccine activity, enhancing immune response, buffering acid and alkali, reducing injection pain and preventing microbial contamination".

[0101] Eighthly, the present invention provides a pharmaceutical composition comprising any of the above-described fusion proteins, nucleic acids, expression vectors, or lipid nanoparticles.

[0102] The pharmaceutical composition further includes pharmaceutically acceptable excipients, including but not limited to: any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

[0103] The administration methods of the pharmaceutical composition include, but are not limited to, oral, injection, implantation, external application, spray, inhalation, or combinations thereof.

[0104] In a ninth aspect, the present invention provides a method for the prevention and / or treatment of a disease, comprising administering to a subject an effective dose of any of the aforementioned fusion proteins, nucleic acids, expression vectors, or lipid nanoparticles.

[0105] The term "subject" includes living organisms (e.g., mammals) that can elicit an immune response. Examples of subjects include humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0106] The terms “treat” or “treatment” or “ameliorate” refer to the medical management of a subject’s disease, condition, or undesirable condition. Treatment or preventative benefits include improved clinical outcomes; reduction or alleviation of symptoms associated with the disease, condition, or undesirable condition; reduced symptom occurrence; improved quality of life; longer disease-free status; reduction in the severity of the disease, condition, or undesirable condition; stabilization of the disease state; delay in disease progression; remission; survival; prolonged survival; or any combination thereof.

[0107] The term "therapeutic effective dose" refers to a pharmaceutically considered effective dosage, that is, an amount of active drug sufficient to significantly improve the condition without causing serious side effects. Dosage depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality, and individual response of the patient or animal, the route of administration, frequency of administration, and therapeutic purpose; therefore, the dosage of this invention can vary widely.

[0108] The present invention has at least the following beneficial effects: 1. Compared with the existing technology, the present invention has better technical effects in terms of low dosage, high safety and long-lasting immune memory.

[0109] According to experimental tests, this invention enables the effective activation of CD8 T cells, which was previously impossible with existing technologies, to be significantly activated.

[0110] 2. Innovative molecular design The design employs a dual antigen of Mpt83 and Hsp65, with single-stranded tandem expression of mRNA to enhance broad-spectrum immunity.

[0111] Adding optimized tPA signal peptide (N-terminus) and MITD sequence (C-terminus) enhances antigen expression and presentation efficiency.

[0112] By optimizing codons and adjusting GC content, mRNA stability and protein expression levels can be improved.

[0113] 3. Excellent immune response It induces highly efficient humoral and cellular immunity, with specific antibody titers reaching 1 million in mouse experiments.

[0114] It significantly activates specific T-cell immune responses, demonstrating good vaccine potential.

[0115] A low dose (1 µg mRNA-LNP) can effectively activate immunity, making it more economical and safer for treating tuberculosis. It is scalable and can be widely applied to the research and development and production of other TB mRNA vaccines. Attached Figure Description

[0116] Figure 1 The results show the detection of serum Mpt83 binding antibody titers in mice during the drug screening stage of Example 1 of this invention.

[0117] Figure 2 The results show the detection of Hsp65 binding antibody titers in the serum of mice in each group during the drug screening stage of Example 1 of this invention.

[0118] Figure 3 The titer of Mpt83-specific binding antibody in the serum of mice immunized with the mRNA-LNP-4 vaccine in Example 2 of this invention was detected.

[0119] Figure 4 The titer of Hsp65-specific binding antibody in the serum of mice immunized with the mRNA-LNP-4 vaccine in Example 2 of this invention was detected.

[0120] Figure 5 This invention was developed to detect antigen-specific CD4+ induced by splenic lymphocytes in mice from each group on day 97 after immunization with the mRNA-4 vaccine in Example 2 of this invention. + and CD8 + Expression levels of T cell subsets IFN-γ and CD69.

[0121] Figure 6 This invention was used to detect IFN secretion by splenic lymphocytes in each group of mice in Example 2 of this study. Results of γ T lymphocyte spot count detection.

[0122] Figure 7 The results show the titers of Mpt83 and Hsp65 specific binding antibodies in Comparative Example 1 of this invention.

[0123] Figure 8 This invention relates to antigen-specific CD4+ induced by mouse spleen lymphocytes in Comparative Example 1 of the present invention. + and CD8 + CD69 expression level of T cell subset.

[0124] In the attached diagram, the key experimental operation days such as vaccine administration and sampling are designated as "Dn day", representing the nth day after the baseline date. This is used to clearly define the experimental timeline and sample collection nodes. For example, "D0" refers to the day of the first vaccine administration in the experiment, which is the core baseline date for all time nodes, and "D1" is the first day after "D0 (administration day)". Detailed Implementation

[0125] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0126] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0127] Plasmid extraction kit, GK2004-200, provided by Shanghai Jereh Biotechnology Co., Ltd.; DNA purification magnetic beads, N411-02-AA, provided by Nanjing Novizan Biotechnology Co., Ltd.; Restriction endonuclease BspQI, R0712S, provided by New England Biotechnology (Beijing) Co., Ltd.; T7 RNA in vitro transcription kit: trade name T7 HighYield RNA Transcription kit, DD4101-PB-01, provided by Nanjing Novizan Biotechnology Co., Ltd.; RNA purification lithium chloride, trade name Lithium Chloride, AM9480, provided by Thermo Fisher Scientific; Capping kit, trade name CAP GAU m7G(5')ppp(5')(2' OMeA)pU, CAP30112-1, provided by Jiangsu Shenji Biotechnology Co., Ltd.; RNA fluorescence detection kit, trade name Quant-iT RiboGreen RNA kit, R11490, provided by Thermo Fisher Scientific. The company provided the following: mice (Hangzhou Academy of Medical Sciences); RPMI 1640 culture medium (trade number 11875093, Thermo Fisher Scientific); Mpt83 and Hsp65 peptides (custom-made by Genscript Biotech); eBioscience™ Cell Stimulation Kit (with protein transport inhibitor, PMA+Ionomycin) (500X), catalog number 00-4975-93, Thermo Fisher Scientific; FastImmune™ Brefeldin A (BFA) Solution (trade number 347688, BD Biosciences, Inc.); Mouse IFN-γ Precoated ELISPOT Kit (trade number 2210005, Dako Biotech Co., Ltd.); PerCP-Cy™ 5.5 Rat Anti-Mouse CD3 (trade number 3138327, BD Biosciences, Inc.); PE / Cyanine7 anti-mouse CD4 Antibody (trade number 116016, BD Biosciences, Inc.); FITC Rat Anti-Mouse CD8a (53-6.7), product number 553030, provided by BD Pharmaceuticals, USA; APC Rat Anti-Mouse IFN-γ (XMG1.2), product number 554413, provided by BD Pharmaceuticals, USA; CD69 Monoclonal Antibody (H1).2F3), PE, product number 12-0691-82, provided by BD Corporation, USA; Cytofix / Cytoperm™ PlusFixation / Permeabilization Solution Kit, product number 555028, provided by BD Corporation, USA; BD Perm / Wash™ Perm / Wash Buffer, product number 554723, provided by BD Corporation, USA.

[0128] Construction of a self-replicating vector in a basic implementation example JJ-SaRNA-2 is a self-replicating RNA vector constructed based on the replicon backbone of Venezuelan equine encephalitis virus (VEEV). Its structure includes: a 5′ untranslated region (5′ UTR); sequences encoding VEEV non-structural proteins nsP1–nsP4 (responsible for RNA replication and transcription); a 26S subgene promoter located downstream of nsP4 (driving efficient expression of exogenous genes); and a 3′ untranslated region (3′ UTR) and a poly(A) tail.

[0129] Example 1: Fusion Protein Molecule Design 1.5' signal peptide design and mutation The preferred tPA signal peptide of this invention has been optimized through mutation to enhance the secretion efficiency of the protein. The signal peptide sequence is shown in SEQ ID NO:1: MDAMKRGLCCVLLLLCGAVFVSASA, and the unmutated signal peptide sequence is shown in SEQ ID NO:2: MDAMKRGLCCVLLLCGAVFVSPSA.

[0130] 2. Molecular design of antigen proteins (1) Selection of target antigen The Mpt83 protein of this invention is used as the target antigen, and its amino acid sequence is shown in SEQ ID NO:3: CSSTKPVSQDTSPKPATSPAAPVTTAAMADPAADLIGRGCAQYAAQNPTGPGSVAGMAQDPVATAASNNPMLSTLTSALSGKLNPDVNLVDTLNGGEYTVFAPTNAAFDKLPAATIDQLKTDAKLLSSILTYHVIAGQASPSRIDGTHQTLQGADLTVIGARDDLMVNNAGLVCGGVHTANATVYMIDTVLMPPAQ.

[0131] The amino acid sequence of the target antigen Hsp65 of this invention is shown in SEQ ID NO:4: VAKKTDDVAGDGTTTATVLAQALVKEGLRNVAAGANPLGLKRGIEKAVDKVTETLLKDAKEVETKEQIAATAAISAGDQSIGDLIAEAMDKVGNEGVITVEESNTFGLQLELTEGMRFDK.

[0132] 3. MITD sequence molecular design The MITD sequence is a special sequence that can be added to antigens in vaccines to guide them into specific regions within dendritic cells. When antigens are guided by signals carrying the MITD sequence, they are more likely to be processed by dendritic cells and presented to T cells, thereby initiating an immune response against them. This invention preferably uses the MITD sequence and designs mutations in it, the sequence being SEQ ID NO:5: LQIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSESQAACSDSAQGSDVSLTA.

[0133] 4. Connector Design The Mpt83 antigen protein and the Hsp65 antigen protein are linked using a flexible linker, the sequence of which is SEQ ID NO:6: GGSGGGSGGGSGGGS; the Hsp65 antigen protein and the MITD sequence are linked using a flexible linker, the sequence of which is SEQ ID NO:7: GGSGGGSGGGS 5. Optimization design of the termination codon To achieve precise control over protein translation, protein synthesis can be terminated by adding a stop codon, thereby producing the desired antigen protein. The stop codon sequence is designed as SEQ ID NO:8: TAATAATGATAG.

[0134] Example 2: Design of Mycobacterium tuberculosis mRNA 1. Designed based on the tandem sequence of the antigen protein, the amino acid sequence is shown in SEQ ID NO:9: MDAMKRGLCCVLLLLCGAVFVSASACSSTKPVSQDTSPKPATSPAAPVTTAAMADPAADLIGRGCAQYAAQNPTGPGSVAGMAQDPVATAASNNPMLSTLTSALSGKLNPDVNLVDTLNGGEYTVFAPTNAAFDKLPAATIDQLKTDAKLLSSILTYHVIAGQASPSRIDGTHQTLQGADLTVIGARDDLMVNNAGLVCGGVHTANATVYMI DTVLMPPAQGGSGGGSGGGSGGGSVAKKTDDVAGDGTTTATVLAQALVKEGLRNVAAGANPLGLKRGIEKAVDKVTETLLKDAKEVETKEQIAATAAISAGDQSIG DLIAEAMDKVGNEGVITVEESNTFGLQLELTEGMRFDKGGSGGGSGGGSLQIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSESQAACSDSAQGSDVSLTA.

[0135] 2. Based on the codon usage preferences of host cells, commonly used synonymous codons in host cells are selected to improve gene expression levels. The stability of mRNA secondary structure is closely related to mRNA half-life and protein expression levels. Enhancing the stability of mRNA secondary structure is beneficial to the expression of target proteins. High levels of guanine and cytosine (G and C) can improve mRNA stability and translation efficiency. Adjusting the GC content in the codon sequence of the target gene can improve its expression level in host cells. The average GC content of the optimized sequence was 57%-65%; after further optimization, the GC content was 59%-64%; the final optimized GC content was determined to be 63%, and the final optimized base sequence is SEQ ID NO:10:

[0136] 3. Construction of mRNA expression plasmids A self-replicating vector (containing elements as described in Basic Example 1) was used as the expression vector. The mRNA antigen-coding sequence (SEQ ID NO: 10) was directionally cloned into the vector's multiple cloning site (MCS) using a restriction endonuclease digestion-ligation method. The mRNA antigen-coding sequence was constructed in the following order: (1) Insertion of human tissue-type plasminogen activator (tPA) signal peptide at the 5' end; (2) The coding sequence of the tandem Mycobacterium tuberculosis antigen Mpt83 (SEQ ID NO:3); (3) Connected via a flexible linker sequence (SEQ ID NO:6); (4) Further insertion of heat shock protein Hsp65 (SEQ ID NO:4); (5) The Hsp65 antigen and the MITD (SEQ ID NO:5) sequence are linked by a flexible linker (SEQ ID NO:7); (6) A stop codon (SEQ ID NO:8) was added to the 3' end to obtain the self-replicating plasmid expression vector for the TB mRNA vaccine. This invention designed four plasmids, which differ in their tPA signal peptide and MITD sequence: Plasmid 1: the signal peptide is the unmutated tPA signal peptide (SEQ ID NO:2), without the MITD sequence; Plasmid 2: the signal peptide is the unmutated tPA signal peptide (SEQ ID NO:1), without the MITD sequence; Plasmid 3: the signal peptide is the unmutated tPA signal peptide (SEQ ID NO:2), with the MITD sequence added (SEQ ID NO:5); Plasmid 4: the signal peptide is the mutated tPA signal peptide (SEQ ID NO:1), with the MITD sequence added (SEQ ID NO:5). The remaining parts have the same structure and design.

[0137] Example 3: mRNA in vitro transcription and lipid nanoparticle encapsulation 1. In vitro transcription of mRNA The supercoiled plasmid was linearized by cutting with the restriction endonuclease BspQI, and the linear plasmid was purified using magnetic beads. Then, it was processed using Nanoparticles. The concentration of linearized plasmids was determined by drop assay, and their digestion efficiency was assessed by gel electrophoresis. A one-step in vitro transcription method was employed, in which the transcription system included T7 RNA polymerase, NTPs, capping enzyme, and capping substrate to achieve simultaneous transcription and capping. After transcription, residual DNA template was digested with DNase I, followed by mRNA purification. mRNA was purified using lithium chloride precipitation, and the concentration and quality of the purified mRNA were determined by gel electrophoresis to evaluate its yield and quality.

[0138] 2. Lipid nanoparticles encapsulating mRNA Ionizable cationic liposomes Dlin-MC3-DMA, distearate phosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) were dissolved in anhydrous ethanol at a molar ratio of 50:7:40:3 and filtered to obtain the organic phase. Four TB-mRNAs were dissolved in citrate buffer at pH 4 to obtain the aqueous phase. The aqueous and organic phases were encapsulated using an Aitesen MPE-L2 microfluidic device at a volume and flow rate ratio of 3:1 to obtain the mRNA-LNP intermediate. The mRNA-LNP intermediate was then ultrafiltered, with the buffer solution replaced by Tris-HCl. The encapsulation efficiency and encapsulated RNA concentration of the mRNA-LNP were then measured using the Quant-iT RiboGreen RNA kit. The electrochemical potential and particle size of the mRNA-LNP were measured using a zeta potential analyzer and a particle size analyzer, respectively. After all indicators met the standards, the mRNA-LNPs were aliquoted and stored at -20°C.

[0139] The particle size of the four mRNA-LNPs was found to be between 98.53 and 111.90 nm, the zeta potential was between 3.87 and -0.13 mV, the polydispersity index was less than 0.15, and the encapsulation efficiency was higher than 94%.

[0140] Table 1 mRNA LNP vaccine quality testing results

[0141] Example 1: Verification and screening of the immunogenicity of 4 mRNA-LNPs in animals. 1. Animal experiments BALB / c mice aged 6-8 weeks were selected and divided into 5 groups of 5 mice each: control group (empty LNP), mRNA-LNP-1, mRNA-LNP-2, mRNA-LNP-3, and mRNA-LNP-4. Except for the control group, the other groups were administered 10 μg of LNP-encapsulated mRNA. All groups received a single dose. Blood samples were collected from the orbital sinus of mice on days 0, 7, 14, 21, and 28 after administration to detect the titers of Mpt83 and Hsp65-specific binding antibodies in serum. Simultaneously, on day 28, spleens from 3 mice were collected for ELISPOT detection of IFN-γ secretion, and mRNAs with high immunogenicity were screened.

[0142] Table 2 Animal experimental grouping and dosing information during the drug screening phase

[0143] 1.1 Detection of binding antibodies in mouse serum Recombinant Mpt83 or Hsp65 proteins were coated onto 96-well plates and incubated overnight at 4 °C. After blocking with BSA, serially diluted mouse serum was added and incubated at 37 °C. After washing with PBST, HRP-labeled goat anti-mouse IgG secondary antibody was added, followed by further incubation and washing. TMB substrate was then added for color development, and the absorbance was measured at 450 nm after terminating the reaction. Antibody titer was expressed as the highest dilution where the OD value exceeded the background by 2.1 times.

[0144] Four mRNAs were encapsulated within LNPs, and mice were immunized with the drugs. The titers of Mpt83 and Hsp65-specific binding antibodies in serum were measured at different time points. Figure 1 and Figure 2 (Legendary letter D represents day.) The results show that the titers of Mpt83 and Hsp65-specific binding antibodies in the LNP-4 vaccine were higher than those in other groups at all time points, and the titer of Mpt83-specific binding antibodies in the LNP-4 vaccine was particularly high on days 21 and 28, reaching 10,000. This indicates that the design of mRNA-4, i.e., the tPA mutant signal peptide plus the MITD sequence, was optimal.

[0145] Example 2: Immunological evaluation of mRNA vaccine dosage. 1. Animal Experiment Design and Sampling BALB / c mice aged 6-8 weeks were selected and observed for one week after acceptance to reduce non-specific stress responses before being grouped for the experiment. Using mRNA-4 screened in Example 1 as the vaccine research subject, the experiment was designed with five groups: control group (empty LNP), 1 μg mRNA-LNP-4 immunization dose group, 2.5 μg mRNA-LNP-4 immunization dose group, 5 μg mRNA-LNP-4 immunization dose group, and 10 μg mRNA-LNP-4 immunization dose group. Mice in all groups were administered the vaccine via intramuscular injection in the hind leg on days 0, 14, and 28, respectively. The control group was injected with 0.05 mL of empty LNP. Observe the animals daily and record clinical symptoms, including but not limited to: 0. Normal; 1. Reduced activity; 2. Restlessness; 3. Trembling; 4. Circling or backing away; 5. Hair pricking; 6. Abnormal breathing; 7. Arched back; 8. Significantly decreased body temperature; 9. Hair loss; 10. Eye abnormalities; 11. Abdominal swelling; 12. Other (describe as needed); NA; Dead / euthanized. Blood samples were collected from the orbital sinuses of mice on days 0, 14, 21, 28, 42, 58, and 97 to detect the titer of binding antibodies in the serum. On day 21, the spleen was harvested from mice, and the secretion of the cytokine IFN-γ was detected using ELISPOT. On day 97, the spleen was harvested from mice, and CD4+ was detected using flow cytometry. + and CD8 + T-cell immune response.

[0146] Observations in mice showed that no obvious abnormal behaviors or physiological responses occurred after administration, and these observations were independent of the dosage. Within the tested dosage groups, mice were able to eat and perform their daily activities normally.

[0147] Table 3. Vaccine Dosage and Immunological Evaluation Mice Grouping and Dosing Information

[0148] 2. Detection results of antigen-specific binding antibodies in the serum of mice immunized with mRNA-LNP-4 vaccine The results of antigen-specific binding antibody detection in immunized mouse serum are as follows: Figure 3 and Figure 4 As shown.

[0149] The results showed that the TB mRNA vaccine immunized mice with high titers of Mpt83 and Hsp65 specific binding antibodies at different time points. The titers reached their highest values ​​on day 42 (D42), after the third immunization, with Mpt83 antigen specific binding antibody levels as high as 1,000,000 and Hsp65 antigen specific binding antibody levels as high as 100,000. There was no difference between different dose groups, that is, the Mpt83 and Hsp65 antigen specific antibody titers were consistent in the 1 μg, 2.5 μg, 5 μg, and 10 μg dose groups, indicating that the dosage of 1 μg was sufficient, and the specific binding antibody levels remained high 69 days after the third immunization.

[0150] 3. Flow cytometry analysis of spleen cells from mice immunized with mRNA-LNP-4 vaccine Single-cell suspensions were prepared from mouse spleens. After erythrocyte lysis and washing, the cell density was adjusted to approximately 4 × 10⁶ cells / mL using complete culture medium. 6 10 cells / mL, dispensed into 96-well U-plates (approximately 2 × 10⁻⁶ cells / mL). 5 Cells were cultured at 37 °C and 5% CO2, and stimulated in vitro with a mixture of Mpt83 and Hsp65 peptides. DMSO served as a blank control. Stimulation lasted approximately 40 h, followed by the addition of BFA to block secretion in the last 16 h of culture. Cells were collected and subjected to sequential staining for viability and dead cells, surface marker staining (e.g., CD3, CD4, CD8, CD69), followed by fixation and permeabilization and intracellular IFN-γ antibody staining. Finally, T cell activation and cytokine expression were detected by flow cytometry.

[0151] Mice were immunized three times with the mRNA vaccine. On day 97, the spleens of the mice were harvested for antigen-specific CD4 assay. + and CD8 + T-cell flow cytometry analysis results are as follows Figure 5 As shown, compared with the empty LNP injection group (control group), the spleen cells of mice injected with different doses of mRNA vaccine showed increased activation of CD69 after peptide stimulation in vitro. + T cells (CD4) + / CD8 + The high proportion of [a specific cell type] indicates that the mRNA vaccine elicited a strong cellular immune response in mice. However, no correlation was shown between the immune response and the administered dose. A 1 μg dose achieved the same cellular immune response as a 10 μg dose in mice, demonstrating that a 1 μg injection of the self-replicating mRNA vaccine of this invention can induce a T-cell immune response in mice. By comparing CD4 [cell type]... + IFN-γ + T occupies a total of CD4 +The T cell ratio indicates that the 1 μg immunization group elicited a strong Th1 immune response, suggesting that a low dose of 1 μg is sufficient to activate the T cell immune response. Furthermore, the sampling and detection time was 69 days after the third immunization for CD4. + and CD8 + The proportion of activated T cells remains high.

[0152] 4. ELISPOT testing (1) Take the spleens of mice from each group in this implementation case, grind them, collect the cell suspension, centrifuge, and wash once with RPMI-1640 medium; (2) Lyse red blood cells: Lyse the spleen cell samples of each mouse with 2 mL of red blood cell lysis buffer for 2 minutes, then terminate the red blood cell lysis with 10 mL of stop solution, centrifuge to collect the cells, resuspend the cells in an appropriate amount of RPMI-1640 medium containing 10% FBS, and then count the cells; (3) Add sterile DPBS to ELISPOT plates pre-coated with IFN-γ, 200 μL / well, blot clean, repeat 4 times. Then add 200 μL of RPMI-1640 medium containing 10% FBS to each well and block at room temperature for 30 minutes, blot clean; (4) Add 2×10 5 One spleen cell and the corresponding antigen peptide (final concentration 10 μg / mL), the blank control group was treated with DMSO, and the positive control group was treated with PMA (final concentration 50 ng / mL) and Ionomycin (final concentration 1 μg / mL). After culturing at 37℃ and 5% CO2 for 40-48 h, spot detection was performed; (5) Discard the cell contents and detect the spots according to the steps in the ELISPOT kit manufacturer's instructions. Finally, the number of spots was calculated using an enzyme-linked spot counter.

[0153] On day 21 (7 days after the second immunization), mouse spleen cells were harvested. A mixture of Mpt83 and Hsp65 peptides was used to stimulate the spleen cells, and the secretion of the cytokine IFN-γ was detected. Results are as follows: Figure 6As shown, PMA+Ionomycin (positive control) treatment nonspecifically stimulated cells to secrete IFN-γ. DMSO treatment (negative control) and Irrelevent peptide treatment (irrelevant peptide) resulted in very low numbers of IFN-γ-positive cells. However, after TB peptide treatment (TB antigenic determinant), the number of antigen-specific IFN-γ-positive cells significantly increased in the 1 μg, 2.5 μg, 5 μg, and 10 μg mRNA vaccine dose groups, exceeding that of the empty LNP injection group. Furthermore, the 1 μg mRNA-LNP4 dose group showed a higher number of IFN-γ-positive cells than other dose groups. These results indicate that the mRNA-4 vaccine encoding Mpt83 and Hsp65 activates a specific T-cell immune response, potentially involving immune memory cells and possibly leading to an inflammatory response, which is crucial for pathogen clearance and immune protection.

[0154] Comparative Example 1: The difference from the previous example is that tPA signal peptide and MITD were not added; instead, Mpt83 (SEQ ID NO:3) and Hsp65 (SEQ ID NO:4) were linked together using a flexible linker sequence (SEQ ID NO:6). Encapsulation was performed as described in Example 3, and the resulting RNA was named TB-mRNA.

[0155] Animal experiments: The experiment consisted of two groups of three mice each. BALB / c mice aged 6-8 weeks were used as the control group (empty LNP) and the experimental group (TB-mRNA). Except for the control group, the experimental group received 15 μg of LNP-coated mRNA. Blood samples were collected from the orbital sinus of mice on days 14 and 28 to detect the titers of Mpt83 and Hsp65-specific binding antibodies in the serum. On day 28, spleens from three mice were collected for flow cytometry to detect T cell activation and assess the immunogenicity of the vaccine. The results are shown below. Figure 7 and Figure 8 .

[0156] Depend on Figure 7 It can be seen that by detecting the specific binding antibody titers of Mpt83 and Hsp65, the binding antibody titers of both are low. After the second immunization, the binding antibody titer of Mpt83 is 1:2000, while the binding antibody titer of Hsp65 is 1:1500.

[0157] Depend on Figure 8 It can be seen that the vaccine group prepared in the comparative proportion failed to effectively induce CD4. ＋ and CD8 ＋ Increased expression of CD69 on the surface of T cells failed to promote T cell activation.

[0158] In summary, the mRNA vaccine carrying the tPA signal peptide, MITD sequence, and dual antigen combination disclosed in this invention demonstrates great potential as a preventive and therapeutic vaccine for tuberculosis by inducing a strong and specific immune response against key antigens of Mycobacterium tuberculosis. This vaccine utilizes self-replicating mRNA technology to optimize antigen expression and immunogenicity, while ensuring safety and efficacy through ultra-low dose control. Furthermore, the long-term immunological memory effect of this vaccine provides a scientific basis for its widespread application in clinical practice. Therefore, the vaccine product of this invention not only holds promise as a new strategy for tuberculosis control but may also contribute significantly to the global elimination of tuberculosis.

[0159] The embodiments described above do not represent the only form of the present invention, and the scope of protection of the present invention covers all improvements and variations that follow its core ideas and principles.

[0160] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A fusion protein, characterized in that, It includes Hsp65 antigen and Mpt83 antigen, tPA signal peptide and MITD sequence.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the Hsp65 antigen is SEQ ID NO.4 or a sequence with greater than 85% homology to SEQ ID NO.4; The amino acid sequence of the Mpt83 antigen is SEQ ID NO.3 or a sequence with greater than 85% homology to SEQ ID NO.3; The amino acid sequence of the tPA signal peptide is SEQ ID NO.1 or a sequence with greater than 85% homology to SEQ ID NO.1; The amino acid sequence of the MITD sequence is SEQ ID NO.5 or a sequence with greater than 85% homology to SEQ ID NO.

5.

3. A nucleic acid, characterized in that, The nucleic acid encodes the fusion protein according to any one of claims 1-2.

4. The nucleic acid according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid is SEQ ID NO.10 or a sequence that has more than 85% homology with SEQ ID NO.

10.

5. An expression carrier, characterized in that, The expression vector expresses the fusion protein according to any one of claims 1-2 or carries the nucleic acid according to any one of claims 3-4.

6. A method for encapsulating lipid nanoparticles, characterized in that, Includes the following steps: The encapsulation material is dissolved in an organic phase, and the fusion protein according to any one of claims 1-2, the nucleic acid according to any one of claims 3-4, or the expression vector according to claim 5 is dissolved in an aqueous phase. Then, the organic phase and the aqueous phase are mixed for encapsulation. The encapsulation material includes at least one of Dlin-MC3-DMA, SM-102, ALC-0315, 306Oi10, 503Oi10, DSPC, DOPE, cholesterol, DMG-PEG2000, PEG2000-PE, and C14-PEG.

7. The lipid nanoparticles prepared by the lipid nanoparticle encapsulation method according to claim 6.

8. The use of the fusion protein according to any one of claims 1-2, the nucleic acid according to any one of claims 3-4, the expression vector according to claim 5, or the lipid nanoparticle according to claim 7 in the preparation of a medicament for the prevention and / or treatment of Mycobacterium tuberculosis-related diseases.

9. A tuberculosis vaccine, characterized in that, It comprises the fusion protein according to any one of claims 1-2, the nucleic acid according to any one of claims 3-4, the expression vector according to claim 5, or the lipid nanoparticles according to claim 7.

10. A pharmaceutical composition, characterized in that, It comprises the fusion protein according to any one of claims 1-2, the nucleic acid according to any one of claims 3-4, the expression vector according to claim 5, or the lipid nanoparticles according to claim 7.

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