SaRNA vaccine for echinococcosis as well as preparation method and application of SaRNA vaccine

Abstract

The invention discloses an SaRNA vaccine for echinococcosis as well as a preparation method and application of the SaRNA vaccine. The preparation method of the SaRNA vaccine comprises the following steps: carrying out codon optimization on a modified target antigen protein through a genetic engineering technology, then assembling the modified target antigen protein with a self-replicating protein sequence, 5 'UTR, 3' UTR and Poly (A) tail, carrying out gene synthesis, then cloning the synthesized gene into a plasmid, and carrying out purification to obtain the SaRNA vaccine. The preparation method comprises the following steps: constructing recombinant plasmids, sequentially carrying out plasmid linearization, in-vitro transcription and purification on the constructed recombinant plasmids to prepare SaRNA molecules, and finally wrapping the SaRNA molecules in lipid nanoparticles to form the SaRNA vaccine for the echinococcosis. Experiments prove that the SaRNA vaccine can activate humoral immunity and cellular immunity of mice at the same time, high-level EG95 specific antibodies and cytokines can be generated through low-dose immunity, and the SaRNA vaccine has wide application prospects in the aspect of preventing and / or treating the echinococcosis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a SaRNA vaccine for echinococcosis, its preparation method, and its application. Background Technology

[0002] Echinococcosis is a serious zoonotic parasitic disease caused by the larvae of the Echinococcus tapeworm, which parasitize the liver, lungs, and other organs of humans and animals. This disease can lead to numerous complex and serious complications in both humans and animals, and in severe cases, threaten the life of the host. It is widely distributed in regions worldwide where livestock farming is prevalent. Currently, the recognized Echinococcus tapeworm (… Echinococcus Echinococcosis (Spp.) comprises at least nine species, each with multiple genotypes. Due to its diverse species, wide distribution, numerous host species, and extensive species / strain variation, monitoring and control of this parasitic disease are increasingly challenging. Current control efforts primarily focus on therapeutic drugs and vaccination. Commonly used drugs include anthelmintics (such as praziquantel), anti-echinococcosis drugs (such as albendazole, mebendazole, and their derivatives), and antitumor drugs (such as tamoxifen and 5-fluorouracil). While all these drugs have significant killing effects on echinococcosis larvae, they suffer from drug resistance, dose dependence, low absorption rates in some cases, and certain side effects. Although canine deworming has some effect, effective vaccines are still needed to support the control of echinococcosis in my country's vast populations of cattle and sheep.

[0003] Existing commercially available echinococcosis vaccines are subunit vaccines. Although widely used, they suffer from low immunity and short duration of immunity, failing to meet current needs for echinococcosis control. Therefore, developing novel vaccines with high efficacy is urgent and important. An ideal echinococcosis vaccine should have high immunity levels, long duration of immunity, and low production costs. Since 2020, mRNA vaccines have gained favor in the scientific community due to their advantages such as convenient design, ease of operation, low cost, and short research cycle. Compared to traditional vaccines, mRNA vaccines have significant advantages, including low cost, high efficacy, and easy availability. SaRNA vaccines (self-amplifying mRNA vaccines) belong to a new generation of mRNA technology. They embed a self-replicating sequence into traditional mRNA. After entering cells, they can utilize host cell mechanisms to amplify the RNA multiple times, then translate it into a large amount of the target antigen protein. Therefore, only extremely low doses are needed to induce an immune response comparable to or even stronger than that of traditional mRNA.

[0004] The research and application of SaRNA vaccines in the prevention and treatment of human diseases are rapidly increasing, but research on veterinary vaccines, especially parasitic vaccines, is scarce. Echinococcosis has shown a persistently high incidence rate in recent years, with an upward trend in new cases. The epidemic situation in key areas remains severe, with complex epidemiological factors and a continuously rising risk of transmission and spillover. Echinococcosis prevention and control still faces serious challenges. Traditional vaccines, characterized by low immunization efficacy, are no longer sufficient for current immunization control of this disease. Therefore, the development of SaRNA vaccines for echinococcosis is crucial. Summary of the Invention

[0005] The purpose of this invention is to provide a SaRNA vaccine for echinococcosis, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention first provides a SaRNA vaccine against echinococcosis.

[0007] The echinococcosis SaRNA vaccine provided by this invention contains a self-replicating mRNA molecule; The self-replicating mRNA molecule contains an mRNA sequence that expresses the target antigen protein; The amino acid sequence of the target antigen protein is shown in Sequence 1.

[0008] Furthermore, the mRNA sequence expressing the target antigen protein is shown in Sequence 2.

[0009] Furthermore, the self-replicating mRNA molecule comprises, from the 5' end to the 3' end, a 5' cap structure, a 5' UTR mRNA sequence, an mRNA sequence expressing the self-replicating protein nsp1-4, an mRNA sequence expressing the target antigen protein, a 3' UTR mRNA sequence, and a 3' polyadenylate tail mRNA sequence.

[0010] In some implementations, the 5' end cap structure is a Cap1 structure.

[0011] In some implementations, the mRNA sequence of the 5'UTR is as shown in Sequence 3.

[0012] In some embodiments, the amino acid sequences of the self-replicating proteins nsp1-4 are shown in Sequence 6.

[0013] In some embodiments, the mRNA sequence of the self-replicating protein nsp1-4 is shown in Sequence 7.

[0014] In some implementations, the mRNA sequence of the 3'UTR is shown in Sequence 4.

[0015] In some embodiments, the mRNA sequence of the 3' polyadenylate tail is shown in Sequence 5.

[0016] In some embodiments, the self-replicating mRNA molecule consists, from the 5' end to the 3' end, a 5' cap structure, a 5' UTR mRNA sequence, an mRNA sequence expressing the self-replicating proteins nsp1-4, a Kozak sequence (GCCACC), an mRNA sequence expressing the target antigen protein, a 3' UTR mRNA sequence, and a 3' polyadenylate tail mRNA sequence.

[0017] Furthermore, the echinococcosis SaRNA vaccine is formed by encapsulating the self-replicating mRNA molecule within lipid nanoparticles.

[0018] In some embodiments, the lipids in the lipid nanoparticles include ionizable lipids, phospholipids (DSPC), PEGylated lipids, and cholesterol (CHOL).

[0019] In some embodiments, the ionizable lipid may be ionizable lipid T19.

[0020] In some embodiments, the PEGylated lipid may be DMG-PEG.

[0021] In some embodiments, the molar ratio of the ionizable lipid T19, the phospholipid DSPC, the PEGylated lipid DMG-PEG, and the cholesterol CHOL may be 50:10:1.5:38.5.

[0022] In some implementations, the particle size of the echinococcosis SaRNA vaccine may be 84-86 nm.

[0023] In some implementations, the particle size of the echinococcosis SaRNA vaccine is 85.41 nm.

[0024] To achieve the above objectives, the present invention also provides any of the following substances: M1) protein, wherein the protein is the aforementioned target antigen protein; M2) is a nucleic acid molecule that encodes the protein described in M1; M3) contains an expression cassette containing the nucleic acid molecule described in M2; M4) A recombinant vector containing the nucleic acid molecule described in M2) or the expression cassette described in M3); M5) A transgenic cell line containing the nucleic acid molecule described in M2), the expression cassette described in M3), or the recombinant vector described in M4; M6) SaRNA molecule, wherein the SaRNA molecule is the self-replicating mRNA molecule described above; M7) Echinococcus infection prevention and treatment preparation, wherein the active ingredient of the Echinococcus infection prevention and treatment preparation is the above-mentioned self-replicating mRNA molecule or a delivery system loading the self-replicating mRNA molecule.

[0025] In the above M2), the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0026] In some embodiments, the nucleic acid molecule is a DNA molecule that has 75% or more identity with positions 7530-8699 of sequence 8 and encodes the target antigen protein.

[0027] In some embodiments, the nucleic acid molecule is sequence 2 or an RNA molecule that has 75% or more identity with sequence 2 and encodes the target antigen protein.

[0028] Those skilled in the art can readily use known methods, such as directed evolution and point mutation, to mutate the nucleotide sequence encoding the target antigen protein of the present invention. Those artificially modified nucleotide sequences encoding the aforementioned target antigen protein that have 75% or higher, or 76% or higher, or 77% or higher, or 78% or higher, or 79% or higher, or 80% or higher, or 81% or higher, or 82% or higher, or 83% or higher, or 84% or higher, or 85% or higher, or 86% or higher, or 87% or higher, or 88% or higher, or 89% or higher, or 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity, as long as they encode the aforementioned target antigen protein and have the same function, are all derived from the nucleotide sequences of the present invention and are equivalent to the sequences of the present invention.

[0029] In the above M3), the expression cassette refers to DNA capable of expressing the target antigen protein in a host cell. This DNA may include not only a promoter to initiate transcription of the target antigen protein, but also a terminator to terminate transcription. In some embodiments, the expression cassette may further include an enhancer sequence.

[0030] In the above M4), the vector can be a plasmid, granule, bacteriophage, or viral vector. The recombinant vector can be a vector containing the above-mentioned nucleic acid molecule or expression cassette constructed using existing expression vectors. In some embodiments, the recombinant vector is a vector obtained by ligating the DNA molecule shown in sequence 8 into an ARCT-154 plasmid carrying the T7 promoter sequence.

[0031] In the above M5), the cell can be a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is a HEK293T cell.

[0032] In the above M7), the delivery system can be any system known to those skilled in the art for the purpose of mRNA molecule delivery, including but not limited to lipid nanoparticle delivery systems, virus-like particle delivery systems, polymer-based delivery systems, peptide-based delivery systems, etc. In some embodiments, the delivery system is a lipid nanoparticle delivery system.

[0033] When preparing the preparation for the prevention and treatment of Echinococcus larvae infection, diluents, solubilizers, buffers, pH adjusters, and other excipients, such as physiological saline, may also be added.

[0034] The echinococcosis infection prevention and treatment agents can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, or by respiratory tract administration, including nasal spray, oral inhalation and other methods.

[0035] When using the aforementioned echinococcosis infection prevention and treatment preparation to prevent and treat echinococcosis infection, an effective amount of the above-mentioned echinococcosis SaRNA vaccine is administered to the subject organism.

[0036] To achieve the above objectives, the present invention also provides any of the following applications: N1) The use of any of the substances described in M1)-M6) above in the preparation of the above-mentioned Echinococcus larvae SaRNA vaccine; N2) The application of the above-mentioned echinococcosis SaRNA vaccine or the above-mentioned substances in the preparation of products for treating echinococcosis infection; N3) The use of the above-mentioned echinococcosis SaRNA vaccine or the above-mentioned substances in the preparation of products for the treatment and / or prevention of echinococcosis.

[0037] Any of the products mentioned above may be vaccines or drugs.

[0038] The echinococcosis described above is a fine-grained echinococcosis.

[0039] The echinococcosis mentioned above is cystic echinococcosis (CE).

[0040] This invention provides a SaRNA vaccine for echinococcosis. The preparation method of this SaRNA vaccine includes the following steps: After codon optimization of the modified target antigen protein (sequence 1) using genetic engineering technology, it is assembled with the expressed self-replicating protein sequence and the 5'UTR, 3'UTR, and Poly(A) tail, followed by gene synthesis. The synthesized gene is then cloned into an ARCT-154 plasmid containing the T7 promoter sequence. The constructed recombinant plasmid is then subjected to plasmid linearization, in vitro transcription, and purification to prepare echinococcosis-specific SaRNA molecules. Finally, the echinococcosis-specific SaRNA molecules are encapsulated within lipid nanoparticles using a microfluidic method to form the echinococcosis SaRNA vaccine. The immunization efficacy was evaluated using BALB / c mice with four different immunization programs, assessing the effects of the SaRNA vaccine on humoral and cellular immunity in mice. The results showed that all four different immunization programs induced high levels of specific antibodies in mice. Antibody levels increased with increasing immunization frequency and dosage. Furthermore, the levels of IL-4, IL-13, IFN-γ, and TNF-α in the immunized group were significantly higher than those in the control group. This indicates that SaRNA vaccine immunization not only stimulates the spleen of mice to produce high levels of cytokines, enhancing cellular immunity, but also generates high levels of antibodies, forming a highly efficient humoral immune response. These results demonstrate that SaRNA vaccine can simultaneously activate humoral and cellular immunity in mice. Low-dose immunization can produce high levels of EG95-specific antibodies and cytokines, which can be maintained for a long time, showing broad application prospects in the prevention and / or treatment of echinococcosis. Attached Figure Description

[0041] Figure 1 This is a map of the ARCT-154-EG95 plasmid.

[0042] Figure 2 Synthesis and large-scale extraction of ARCT-154-EG95 plasmid. a) Agarose gel electrophoresis of ARCT-154-EG95 plasmid after synthesis. b) Agarose gel electrophoresis of ARCT-154-EG95 plasmid after large-scale extraction.

[0043] Figure 3 This is a schematic diagram of the in vitro transcription and expression process of Echinococcus larvae-specific SaRNA molecules.

[0044] Figure 4 To detect the integrity of Echinococcus larvae-specific SaRNA molecules using capillary electrophoresis.

[0045] Figure 5 To determine the uniqueness of Echinococcus larvae-specific SaRNA molecules in formaldehyde denaturing gel assays.

[0046] Figure 6The target protein was detected after HEK293T cells were transfected with Echinococcus granulosus-specific SaRNA molecules.

[0047] Figure 7 Particle size determination of SaRNA-LNP lipid nanoparticles. a) Particle size determination. b) PDI (dispersion index). Three different colors represent three replicates.

[0048] Figure 8 EG95-specific antibody levels were measured using ELISA. a) EG95 antibody level 14 days after the first immunization. b) EG95 antibody level 14 days after the second immunization. c) EG95 antibody level 30 days after the second immunization. d) EG95 antibody level 60 days after the second immunization.

[0049] Figure 9 The levels of cytokines in each group of mice were detected by ELISA. a) Mouse IFN-γ level; b) Mouse TNF-α level; c) Mouse IL-4 level; d) Mouse IL-13 level. Detailed Implementation

[0050] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0052] The 4-6 week old BALB / c female mice in the following examples were obtained from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, and were housed in an SPF-grade experimental animal center.

[0053] All animal experiments described below were reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of the Chinese Academy of Agricultural Sciences.

[0054] The HEK293T cells used in the following examples are products of the Cell Bank of the Chinese Academy of Sciences.

[0055] The anti-EG95 mouse monoclonal antibody in the following examples is described in the literature "Li Lingling. Preparation and preliminary application of monoclonal antibody against Echinococcus EG95 protein [D]. Chinese Academy of Agricultural Sciences, 2022. DOI:10.27630 / d.cnki.gznky.2022.000486.".

[0056] The EG95 antigen and its preparation method in the following examples are described in the invention patent with authorization announcement number "CN102731641B" and invention title "Modification of Echinococcus granulosus EG95 protein and its expression in yeast".

[0057] The ARCT-154 plasmid used in the following examples is a product of Wuhan HanHai New Enzyme Biotechnology Co., Ltd.

[0058] The DMEM basic medium, 1640 basic medium, FBS, Penstrep antibiotics and trypsin used in the following examples are all products of Gibco.

[0059] The DH5α competent cells, plasmid extraction kit, endotoxin-free plasmid small-volume extraction kit, erythrocyte lysis buffer, and RAPI neutral lysis buffer used in the following examples are all products of Tiangen Biotech Co., Ltd.

[0060] The TMB substrate solution used in the following examples is a product of Surmodics.

[0061] The pH of the PBS solution in the following examples is 7.4.

[0062] The Co-capping T7 in vitro transcription reagent (N1-Me-pUTP, CAP GAG (3'Ome)) (HBP001509) and BspQⅠ restriction endonuclease (HBP001406) used in the following examples are both products of Wuhan HanHai New Enzyme Biotechnology Co., Ltd.

[0063] The CALNP™ mRNA in vitro mRNA transfection reagent used in the following examples is a product of Beijing Dona Pharmaceutical Technology Co., Ltd.

[0064] Example 1: Preparation of Echinococcus granulosus SaRNA vaccine This invention involves codon optimization of the coding sequence of the modified target antigen protein EG95, followed by assembly with the expressed self-replicating protein sequence and the 5'UTR, 3'UTR, and Poly(A) tail. Gene synthesis is then performed, and the synthesized gene fragment is cloned into a plasmid containing the T7 promoter sequence. The constructed recombinant plasmid is subsequently linearized, transcribed in vitro, and capped to prepare SaRNA molecules. Finally, the prepared SaRNA molecules are encapsulated within lipid nanoparticles using a microfluidic method to obtain an echinococcosis SaRNA vaccine. The specific preparation method is as follows: I. Design of Echinococcus granulosus-specific SaRNA molecules The Echinococcus larva-specific SaRNA molecule designed in this invention consists of, from the 5' end to the 3' end, a 5' cap structure, an mRNA sequence of the 5' untranslated region (5'UTR), an mRNA sequence expressing the self-replicating protein nsp1-4, a Kozak sequence, an mRNA sequence expressing the target antigen protein, an mRNA sequence of the 3' untranslated region (3'UTR), and an mRNA sequence of the 3' poly(A) tail.

[0065] The 5' end cap structure is a Cap1 structure.

[0066] The mRNA sequence of the 5' untranslated region (5'UTR) is shown in Sequence 3.

[0067] The mRNA sequence expressing the self-replicating protein nsp1-4 is shown in Sequence 7, and the amino acid sequence of the self-replicating protein nsp1-4 is shown in Sequence 6.

[0068] Kozak sequence: GCCACC.

[0069] The mRNA sequence expressing the target antigen protein is shown in Sequence 2, and the amino acid sequence of the target antigen protein is shown in Sequence 1.

[0070] The mRNA sequence of the 3' untranslated region (3'UTR) is shown in Sequence 4.

[0071] The poly(A) tail mRNA sequence is shown in Sequence 5.

[0072] II. Preparation of Echinococcus granulosus-specific SaRNA molecules 1. Plasmid construction The coding sequence (Sequence 8) of the Echinococcus larva-specific SaRNA molecule in step one was artificially synthesized and cloned into the ARCT-154 plasmid containing the T7 promoter sequence. After transformation into DH5α competent cells, a large number of plasmids were extracted. The extracted plasmids were found to be consistent with the synthesized plasmids. Figure 2 a) The plasmid was digested with BspQⅠ restriction endonuclease, and the integrity of the plasmid was detected by agarose gel electrophoresis. Figure 2 (b) The plasmid that was verified to be correct was named recombinant plasmid ARCT154-EG95. The image of recombinant plasmid ARCT154-EG95 is shown below. Figure 1 As shown.

[0073] 2. In vitro transcription In vitro transcription process such as Figure 3As shown, the specific steps are as follows: The recombinant plasmid ARCT154-EG95 constructed in step 1 was transformed into DH5α competent cells. After a large amount of plasmid extraction, it was digested with BspQⅠ restriction endonuclease to obtain the digested plasmid. Then, using the Co-capping T7 in vitro transcription reagent (N1-Me-pUTP, CAP GAG (3'Ome)) mRNA in vitro transcription kit, in vitro transcription was performed using a one-step capping co-transcription method to obtain SaRNA molecules. The SaRNA molecules were purified and recovered by LiCl precipitation. 1 μL of SaRNA molecules were diluted, denatured at 65℃, and then subjected to capillary electrophoresis. Figure 4 ) and formaldehyde-modified adhesive ( Figure 5 The integrity and uniqueness of the saRNA were detected separately. The results met the requirements for subsequent experiments.

[0074] 3. Transfect HEK293T cells to express proteins The SaRNA molecules prepared in step 2 were transfected into HEK293T cells using the CALNP™ mRNA in vitro mRNA transfection reagent. Cells were collected at 24h, 48h, and 72h after transfection. Total protein was extracted after cell lysis with RAPI, and the expression level of the target protein was detected by Western blot. Figure 6 ).

[0075] III. Preparation and Physicochemical Properties of SaRNA-LNP Lipid Nanoparticles 1. Preparation of SaRNA-LNP lipid nanoparticles The SaRNA molecules prepared in step two were encapsulated within lipid nanoparticles (ionizable lipid T19: phospholipid DSPC: PEGylated lipid DMG-PEG: cholesterol CHOL = 50:10:1.5:38.5 (molar ratio)) by Wuhan HanHai New Enzyme Biotechnology Co., Ltd. using a microfluidic LNP preparation instrument (Nazhida (Shanghai) Nanotechnology Co., Ltd.) to form SaRNA-LNP lipid nanoparticles. These SaRNA-LNP lipid nanoparticles are the Echinococcus granulosus SaRNA vaccine of this application.

[0076] 2. Detection of particle size and dispersion coefficient of SaRNA-LNP lipid nanoparticles The particle size and dispersion index (PDI) of SaRNA-LNP lipid nanoparticles were determined using Malvern Zetasizer Pro (Malvern PANalytical).

[0077] The results showed that the average particle size of the SaRNA-LNP lipid nanoparticles was 85.41 nm, and the dispersion index (PDI) was <0.1, which was in line with expectations. Figure 7 ).

[0078] Example 2: Detection of SaRNA-LNP vaccine-induced immune response in mice Sixty 4-6 week old female BALB / c mice were randomly divided into 6 groups, with 10 mice in each group. The treatment methods for each group are as follows: SaRNA-LNP 2μg secondary immunization group: Each mouse was immunized twice, with a 2-week interval between the first and second immunizations. The doses for both immunizations were the same, and the immunization method was injection. Each injection consisted of 100μL of a solution containing 2μg of Echinococcus granulosus SaRNA vaccine (solvent was PBS solution).

[0079] SaRNA-LNP 10μg secondary immunization group: Each mouse was immunized twice, with a 2-week interval between the first and second immunizations. The doses for both immunizations were the same, and the immunization method was injection. Each injection consisted of 100μL of a solution containing 10μg of Echinococcus granulosus SaRNA vaccine (solvent was PBS solution).

[0080] SaRNA-LNP 20μg single immunization group: Each mouse was immunized once by injection, with each injection consisting of 100μL of a solution containing 20μg of Echinococcus granulosus SaRNA vaccine (solvent was PBS solution).

[0081] SaRNA-LNP 20μg secondary immunization group: Each mouse was immunized twice, with a 2-week interval between the first and second immunizations. The doses for both immunizations were the same, and the immunization method was injection. Each injection consisted of 100μL of a solution containing 20μg of Echinococcus granulosus SaRNA vaccine (solvent was PBS solution).

[0082] CV (Commercially Vaccine) group: Each mouse was immunized twice, with a two-week interval between the first and second immunizations. The doses for both immunizations were the same, and the immunization method was by injection. Each injection consisted of 100 μL of a 20 μg solution containing the EG95 protein of the echinococcosis gene-engineered subunit vaccine (Chongqing Aolong Biological Products Co., Ltd.) (solvent was PBS solution).

[0083] PBS group: Each mouse was immunized twice, with a two-week interval between the first and second immunizations. The doses for both immunizations were the same, and the immunization method was injection, with 100 μL of PBS solution injected each time.

[0084] Blood was collected from the orbital venous plexus of mice two weeks after each immunization, and serum was collected and stored at -80℃. ELISA was used to detect specific antibodies in the serum of immunized mice. ELISA results showed that the EG95-specific antibody level in the experimental groups was significantly higher than that in the CV and PBS groups, and the antibody level increased with increasing immunization dose and number of immunizations. After the second booster immunization, the specific antibody levels in each experimental group were significantly higher than those after the first immunization. Thirty days after the second immunization, the EG95 antibody levels in the 2μg, 10μg, and 20μg booster immunization groups showed a significant upward trend compared to the first immunization, and then a downward trend at 60 days after the second immunization. Furthermore, the EG95-specific antibody level in the 10μg booster immunization group was significantly higher than that in the CV group at 60 days after the second immunization, while the EG95-specific antibody level in the 2μg booster immunization group was comparable to that in the CV group at 60 days after the second immunization. Figure 8 The results show that SaRNA-LNP can induce high levels of specific antibodies in mice at low doses, and the antibody levels increase with the number of immunizations, demonstrating a significant immune advantage compared to the positive control group (CV).

[0085] Thirty days after the second immunization, three mice were randomly selected. Their spleens were dissected and placed in a 70 μm cell sieve, ensuring the spleen tissue remained immersed in Hanks' solution. The spleen tissue was then ground using the black tip of a syringe to prepare a single-cell suspension. The cells were then arranged at a ratio of 1 × 102 6 Cells were seeded in 96-well plates. 72 hours after EG95 antigen stimulation, cell culture supernatant was collected, and the secretion levels of TNF-α, IFN-γ, IL-4, and IL-13 cytokines in spleen cells were detected using ELISA. ELISA results showed that the secretion levels of TNF-α, IFN-γ, IL-4, and IL-13 cytokines in mice immunized with SaRNA-LNP were significantly higher than those in the PBS group, and the cytokine levels in mice immunized a second time with 20 μg were significantly higher than those in the CV group. Figure 9 ).

[0086] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A SaRNA vaccine for echinococcosis, characterized in that: The echinococcosis SaRNA vaccine contains a self-replicating mRNA molecule; The self-replicating mRNA molecule contains an mRNA sequence that expresses the target antigen protein; The amino acid sequence of the target antigen protein is shown in Sequence 1.

2. The echinococcosis SaRNA vaccine according to claim 1, characterized in that: The mRNA sequence expressing the target antigen protein is shown in Sequence 2.

3. The echinococcosis SaRNA vaccine according to claim 1 or 2, characterized in that: The self-replicating mRNA molecule comprises, from the 5' end to the 3' end, a 5' cap structure, a 5' UTR mRNA sequence, an mRNA sequence expressing the self-replicating protein nsp1-4, an mRNA sequence expressing the target antigen protein, a 3' UTR mRNA sequence, and a 3' polyadenylate tail mRNA sequence.

4. The echinococcosis SaRNA vaccine according to any one of claims 1-3, characterized in that: The mRNA sequence of the 5'UTR is shown in Sequence 3.

5. The echinococcosis SaRNA vaccine according to any one of claims 1-4, characterized in that: The amino acid sequences of the self-replicating proteins nsp1-4 are shown in Sequence 6; Alternatively, the mRNA sequence expressing the self-replicating protein nsp1-4 is shown in Sequence 7.

6. The echinococcosis SaRNA vaccine according to any one of claims 1-5, characterized in that: The mRNA sequence of the 3'UTR is shown in Sequence 4.

7. The echinococcosis SaRNA vaccine according to any one of claims 1-6, characterized in that: The mRNA sequence of the 3' polyadenylate tail is shown in Sequence 5.

8. The echinococcosis SaRNA vaccine according to any one of claims 1-7, characterized in that: The echinococcosis SaRNA vaccine is formed by encapsulating the self-replicating mRNA molecule within lipid nanoparticles.

9. Any of the following substances: M1) protein, wherein the protein is any of the target antigen proteins described in claims 1-8; M2) is a nucleic acid molecule that encodes the protein described in M1; M3) contains an expression cassette containing the nucleic acid molecule described in M2; M4) A recombinant vector containing the nucleic acid molecule described in M2) or the expression cassette described in M3); M5) A transgenic cell line containing the nucleic acid molecule described in M2), the expression cassette described in M3), or the recombinant vector described in M4; M6) SaRNA molecule, wherein the SaRNA molecule is any of the self-replicating mRNA molecules described in claims 1-8; M7) Echinococcus infection prevention and treatment preparation, wherein the active ingredient of the Echinococcus infection prevention and treatment preparation is any of the self-replicating mRNA molecules described in claims 1-8 or a delivery system loading the self-replicating mRNA molecules.

10. Any of the following applications: N1) The use of any of the substances described in claims 9 (M1)-M6) in the preparation of the echinococcosis SaRNA vaccine according to any of claims 1-8; N2) The use of the echinococcosis SaRNA vaccine according to any one of claims 1-8 or the substance according to claim 9 in the preparation of products for treating echinococcosis infection; N3) The use of the echinococcosis SaRNA vaccine according to any one of claims 1-8 or the substance according to claim 9 in the preparation of products for the treatment and / or prevention of echinococcosis.

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