A protein of echinococcus Eg95, nucleotide and mRNA encoding the protein

Abstract

The present application relates to a kind of echinococcus Eg95 protein, nucleotide and mRNA for coding the protein of the present application.By optimizing echinococcus Eg95 protein amino acid and nucleotide sequence, echinococcus circular RNA vaccine, echinococcus linear mRNA vaccine and echinococcus self-replicating mRNA vaccine are prepared, the vaccine can provide strong immune protection to 4-month-old lambs only one needle immunization, so that it can still obtain more than 80% protection (higher than 70% required by Ministry of Agriculture and Rural Affairs) on the 180th day after immunization, solve the problem that commercial sheep echinococcosis genetic engineering subunit vaccine immunity is weak, and frequent vaccination is needed to obtain immune protection, reduce the repeated burden of vaccine procurement and labor cost, reduce the stress response to sheep flock, improve vaccination coverage and compliance, and be conducive to the prevention and control of our country hydatid disease.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an Echinococcus granulosus Eg95 protein, the nucleotides encoding the protein, and the mRNA. Background Technology

[0002] Echinococcosis is a serious zoonotic parasitic disease. Currently, the sheep echinococcosis (hydatid disease) genetically engineered subunit vaccine has been approved for marketing in China. When the vaccine is first administered, two immunizations are required in week 0 and week 4 to provide sufficient immune protection for the sheep flock ([1] Wang Shufang, He Jingui. Detection of the immunization effect of sheep echinococcosis genetically engineered subunit vaccine [J]. Chinese Journal of Veterinary Medicine, 2019(3):2.). In areas with high incidence of echinococcosis, the sheep flocks are geographically dispersed. Frequent vaccination leads to repeated expenditures on vaccine procurement and labor costs, and a large stress response in the sheep flock, resulting in a decrease in vaccination coverage and compliance, which is not conducive to the prevention and control of echinococcosis.

[0003] Therefore, developing a echinococcosis vaccine that provides potent immune protection with a single immunization is of great significance for the prevention and control of echinococcosis. Current research indicates that controlling the prevalence of echinococcosis mainly involves interrupting the developmental stages of the Echinococcus tapeworm, controlling infection of intermediate hosts such as humans and animals, preventing or treating definitive hosts such as dogs, and blocking the widespread dissemination of eggs. Vaccination of intermediate hosts can effectively control the prevalence of Echinococcus granulosus. EG95 is one of the natural oncocytosome antigens and is the most effective protective antigen. A vaccine against ovine Echinococcus granulosus has been successfully developed targeting this protein. Patent CN108066755B discloses a modified EG95 amino acid sequence that can significantly reduce the production cost of sheep echinococcosis antigen, greatly simplify the production process, and has many advantages such as safety, high efficiency, and low cost. Patent CN117467683A discloses a recombinant subunit fusion protein rEG95-Fc and its preparation method, using a mutant of the heavy chain constant region fragment of sheep IgG as an immune-enhancing sequence to significantly enhance the duration of immunity. Patent CN115873888A discloses the GS115 yeast transformant of a truncated EG95 protein fragment gene, and the EG95 antigen protein genetically engineered subunit vaccine prepared from it has good immunogenicity. However, the vaccines in the above patents are all administered through two immunizations, and there is no antibody data 180 days after a single immunization, so it is still impossible to achieve the goal of achieving more than 70% protection on day 180 after a single immunization. In addition, no patents or research papers on Echinococcus granulosus mRNA vaccines have been found.

[0004] Therefore, there is an urgent need for a echinococcosis mRNA vaccine that can provide strong immune protection with a single immunization, and whose protective efficacy reaches more than 70% on day 180 after immunization. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of existing technologies and provide a *Echinococcus granulosus* Eg95 protein, the nucleotides encoding this protein, and its mRNA. This invention provides a modified *Echinococcus granulosus* Eg95 protein and the nucleotides encoding this protein, and constructs and prepares mRNA molecules (including circular RNA molecules, linear mRNA, and self-replicating mRNA) containing these nucleotides and an mRNA vaccine. This *Echinococcus granulosus* mRNA vaccine provides potent immune protection with only a single immunization, achieving a protective efficacy of over 80% on day 180 post-immunization, exceeding the 70% requirement of the Ministry of Agriculture and Rural Affairs. This addresses the industry pain point of existing commercial vaccines having weak immunity and requiring frequent vaccinations.

[0006] The objective of this invention can be achieved through the following methods:

[0007] In a first aspect, the present invention provides a modified Echinococcus granulosus Eg95 protein, wherein the modified Echinococcus granulosus Eg95 protein is obtained by linking an immunostimulating sequence to the C-terminus of an Echinococcus granulosus Eg95 protein mutant via a linker arm.

[0008] As one embodiment of the present invention, the amino acid sequence of the Echinococcus granulosus Eg95 protein is shown in SEQ ID NO.14; the Echinococcus granulosus Eg95 protein mutant is one of the following mutations: E at position 52 is mutated to K / E at position 93 is mutated to K, E at position 52 is mutated to K / E at position 93 is mutated to K / P at position 123 is mutated to Q.

[0009] As one embodiment of the present invention, the sequence of the connecting arm is one of SEQ ID NO.15 and SEQ ID NO.16; the immune enhancement sequence is one of SEQ ID NO.17 and SEQ ID NO.18.

[0010] As one embodiment of the present invention, the amino acid sequence of the modified Echinococcus granulosus Eg95 protein is one of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, and the nucleotide sequence encoding the modified Echinococcus granulosus Eg95 protein is one of SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7.

[0011] Preferably, the amino acid sequence of the modified Echinococcus granulosus Eg95 protein is SEQ ID NO.3, and the nucleotide sequence encoding the modified Echinococcus granulosus Eg95 protein is SEQ ID NO.7.

[0012] Secondly, the present invention provides a circular RNA molecule encoding a modified Echinococcus granulosus Eg95 protein, wherein the nucleotide sequence of the circular RNA molecule is one of SEQ ID NO.8, SEQ ID NO.12, and SEQ ID NO.13.

[0013] Preferably, the nucleotide sequence of the circular RNA molecule is SEQ ID NO.8.

[0014] Thirdly, the present invention provides a linear mRNA encoding a modified Echinococcus granulosus Eg95 protein, the nucleotide sequence of which is shown in SEQ ID NO.9.

[0015] Fourthly, the present invention provides a self-replicating mRNA encoding a modified Echinococcus granulosus Eg95 protein, the nucleotide sequence of which is shown in SEQ ID NO.10.

[0016] Fifthly, the present invention provides the application of a modified Echinococcus granulosus Eg95 protein, a circular RNA molecule encoding the modified Echinococcus granulosus Eg95 protein, a linear mRNA encoding the modified Echinococcus granulosus Eg95 protein, or a self-replicating mRNA encoding the modified Echinococcus granulosus Eg95 protein in the preparation of recombinant protein antigens or genetically engineered vaccines.

[0017] The genetically engineered vaccine includes a nucleic acid vaccine; the nucleic acid vaccine includes a DNA vaccine, a circular RNA vaccine, a linear RNA vaccine, and a self-replicating RNA vaccine.

[0018] In a sixth aspect, the present invention provides a genetically engineered vaccine, which is prepared by mixing circular RNA molecules, linear mRNA or self-replicating mRNA with liposomes.

[0019] The preparation process of circular RNA, linear mRNA or self-replicating mRNA and their vaccines in this invention is as follows: (1) gene synthesis and gene cloning; (2) bacterial culture and plasmid extraction and identification; (3) linearized plasmid is transcribed in vitro to form precursor RNA, and the precursor RNA is circularized to form circular RNA; or linearized plasmid is transcribed in vitro and capped and tailed to form linear mRNA or self-replicating mRNA; (4) circular RNA or linear mRNA or self-replicating mRNA is mixed with lipid solution in a microfluidic device to prepare circular RNA vaccine or linear mRNA vaccine or self-replicating mRNA vaccine.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. This invention optimizes the amino acid sequence of Echinococcus granulosus Eg95 protein by mutating specific sites and linking a special immune-enhancing sequence to the C-terminus via a linker arm. Compared with conventional site mutation and conventional immune-enhancing sequence optimization, the prepared vaccine has higher protective efficacy, especially with a protective efficacy of over 80% after 180 days of immunization.

[0022] 2. This invention optimizes the amino acid and nucleotide sequences of the Eg95 protein of Echinococcus granulosus to prepare Echinococcus granulosus circular RNA vaccines, linear mRNA vaccines, and self-replicating mRNA vaccines. This solves the problem of weak immunity and the need for frequent vaccinations to achieve immune protection in commercially available genetically engineered subunit vaccines for sheep echinococcosis (hydatid disease). Furthermore, the Echinococcus granulosus circular RNA vaccine, linear mRNA vaccine, and self-replicating mRNA vaccine of this invention provide over 80% protection (higher than the 70% requirement of the Ministry of Agriculture and Rural Affairs) in 4-month-old lambs after a single immunization, while commercially available vaccines provide 0% protection after a single immunization within 180 days.

[0023] 3. The Echinococcus granulosus circular RNA vaccine of this invention successfully induced the production of long-lived plasma cells specific to the Eg95 protein, which may be one of the important mechanisms for achieving highly effective protection.

[0024] 4. The Echinococcus circular RNA vaccine, Echinococcus linear mRNA vaccine, and Echinococcus self-replicating mRNA vaccine prepared by this invention reduce the frequency of immunization, reduce the burden of repeated expenditures on vaccine procurement and labor costs, reduce stress response in sheep flocks, improve vaccination coverage and compliance, and are beneficial to the prevention and control of echinococcosis. Attached Figure Description

[0025] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0026] Figure 1 This is a schematic diagram of the design of the Eg95 protein antigen from Echinococcus granulosus in Example 1;

[0027] Figure 2 The results of capillary gel electrophoresis of Echinococcus granulosus circular RNA in Example 3;

[0028] Figure 3 The results of Western blot analysis of cells transfected with the Echinococcus granulosus circular RNA vaccine in Example 4;

[0029] Figure 4 The percentage of long-lived plasma cells in the bone marrow cells of mice immunized with the Echinococcus granulosus circular RNA vaccine in Example 6;

[0030] Figure 5The results of Western blot analysis are shown for cells transfected with the linear mRNA vaccine and the self-replicating mRNA vaccine of Echinococcus granulosus in Example 7. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0032] 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.

[0033] Example 1: Construction of gene template plasmid

[0034] The full-length Eg95 gene sequence (GenBank: AY421719.1) was downloaded from GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ), yielding the Eg95 protein amino acid sequence (SEQ ID NO.14). Further, mutants of the Eg95 protein were constructed, and different immunostimulatory sequences were ligated to the C-terminus via linkers (SEQ ID NO.15: GGGGS linker 1 or SEQ ID NO.16: GGGGSGGGGS linker 2) to obtain the amino acid sequences of Eg95-1 (see SEQ ID NO.1), Eg95-2 (see SEQ ID NO.2), and Eg95-3 (see SEQ ID NO.3). Antigen protein design is described in [link to antigen design]. Figure 1 (The sequences of immune enhancement sequence 1 and immune enhancement sequence 2 are shown in SEQ ID NO. 17-18, respectively).

[0035] Using sheep as the expression host, codon optimization was performed on the gene sequences of Eg95, Eg95-1, Eg95-2, and Eg95-3 proteins. This included replacing rare codons containing the word "sheep" in the sequences, modifying the sequences to appropriately increase the content of codons with a third base of G or C while avoiding specific restriction enzyme sites such as EcoRI, BsaI, and XbaI, as well as unfavorable motifs, and ensuring the balance of GC distribution throughout the sequences. The resulting nucleotide sequences were: Eg95 (see SEQ ID NO.4), Eg95-1 (see SEQ ID NO.5), Eg95-2 (see SEQ ID NO.6), and Eg95-3 (see SEQ ID NO.7).

[0036] Referring to patent CN119462855B, a DNA vector for generating circular RNA was constructed. The gene fragment, consisting of the restriction enzyme EcoRI, T7 promoter, intron fragment II, translation initiation element truncation fragment II, Eg95 nucleotide sequence or Eg95-1 nucleotide sequence or Eg95-2 nucleotide sequence or Eg95-3 nucleotide sequence, polyAC, translation initiation element truncation fragment I, intron fragment I, and restriction enzyme BsaI, was sequentially ligated into the pUC57 vector. This was then double-digested with EcoRI and BsaI. Gene synthesis and cloning were outsourced to Nanjing GenScript Biotech Co., Ltd. Finally, the gene was transformed into *E. coli* DH5α, and sequencing confirmed its correctness.

[0037] Example 2: Escherichia coli fermentation and preparation of recombinant plasmids

[0038] The method of Example 4 of prior patent CN117305328A was used to carry out Escherichia coli fermentation, recombinant plasmid preparation and testing.

[0039] Four E. coli strains containing template plasmids (numbered Eg95, Eg95-1, Eg95-2, and Eg95-3) were fermented in a 5L fermenter, and the wet weights of the bacterial cells obtained by centrifugation were 952g, 1021g, 923g, and 901g, respectively.

[0040] Two hundred grams of *E. coli* were taken from each sample and prepared using a standard plasmid preparation procedure. The plasmids underwent cell lysis, ultrafiltration concentration, particle exchange chromatography, gel filtration chromatography, ultrafiltration, and percolation to obtain four template plasmid DNAs (numbered Eg95, Eg95-1, Eg95-2, and Eg95-3), with masses of 118 mg, 109 mg, 104 mg, and 117 mg, respectively. The A260 / A280 ratio was between 1.8 and 2.0. Agarose gel electrophoresis confirmed that the supercoiled ratio was not less than 90%. The prepared recombinant plasmid DNAs met the requirements, as shown in Table 1.

[0041] Table 1 Plasmid DNA Preparation

[0042]

[0043] Example 3: Preparation and Analysis of Circular RNA

[0044] The circular RNA was prepared according to the method in Example 4 of prior patent CN117305328A, as follows:

[0045] (1) Plasmid linearization and purification

[0046] The recombinant plasmid was digested and linearized using the restriction endonuclease BsaI (nearshore protein). The digestion system was as follows: 10× buffer: 2 ml, plasmid: 100 mg, BsaI: 3 ml, water to a final volume of 20 ml, digested overnight at 37°C. After digestion, the linearized plasmid was purified by chromatography using a Capto Q ImpRes (Source 30 Q) column (Cytiva).

[0047] (2) In vitro transcription: The transcription system was prepared as shown in Table 2 below:

[0048] Table 2 In vitro transcription reaction system

[0049]

[0050] In vitro transcription conditions: linearized plasmid template concentration 50 mg, final GTP / ATP / CTP / UTP concentrations 16 mM / 32 mM / 8 mM / 8 mM, final T7 RNA polymerase concentration 1000 KU, incubation at 37℃ / 220 rpm for 2 h, followed by addition of 100 ml deoxyribonuclease I (1000 U / ml) to the transcription system, and incubation at 37℃ / 220 rpm for 15 min.

[0051] (3) Transcription product mRNA precipitation: Add LiCl solution to the above transcription product to a final concentration of 1M and place at -20℃ overnight for precipitation; centrifuge the overnight treatment solution (centrifuge at 16,000 g for 20 minutes at 4°C) to obtain mRNA precipitate, dry at room temperature and resuspend the precipitate in enzyme-free water to obtain linear mRNA aqueous solution (i.e. precursor RNA).

[0052] (4) mRNA cyclization and concentration: The cyclization reaction system is shown in Table 3 below:

[0053] Table 3 In vitro cyclization reaction system

[0054]

[0055] The above solutions were thoroughly mixed. The cyclization conditions were as follows: linear mRNA feed amount 3.5 g, final magnesium ion concentration 8 mM, cyclization reaction temperature 52℃, and cyclization reaction time 15 min. The cyclization reaction products were purified by HPLC (reference doi: 10.3390 / chemosensors12070120), and the target circular RNA molecules were collected, with yields of 21.5 g, 23.2 g, 19.4 g, and 25.3 g, respectively.

[0056] (5) Concentration and analysis of purified circular RNA

[0057] The circular RNA molecules Eg95, Eg95-1, Eg95-2, and Eg95-3 were concentrated using TFF capsules (LVCentramate, PALL), with a membrane area of ​​0.02 m². 2 The flow rate was 120 mL / min. The ultrafiltered liquid was then transferred to a sterile sample bottle after sterile filtration.

[0058] Capillary gel electrophoresis results of four circular RNA molecules are shown below. Figure 2 The sizes are correct and the purity is above 95%, meeting the requirements. The nucleotide sequences of the Eg95-3 circular RNA molecules are shown in SEQ ID NO.8, the Eg95 circular RNA molecules are shown in SEQ ID NO.11, the Eg95-1 circular RNA molecules are shown in SEQ ID NO.12, and the Eg95-2 circular RNA molecules are shown in SEQ ID NO.13.

[0059] Example 4 Preparation and Analysis of Lipid Nanoparticles (LNPs)

[0060] mRNA nanoliposomes were prepared using an LNP encapsulation kit (Nexstar, catalog number: N01001050). Four circular RNA molecules (Eg95, Eg95-1, Eg95-2, and Eg95-3) were diluted with citrate-sodium citrate solution (pH 4.0) (buffer phase) to a concentration of 100 μg / mL. The diluted RNA was then mixed with liposomes at a specific ratio using an LNP microfluidic preparation system (Nexstar, model: NEXSTARnano1) to prepare LNPs (LNP-Eg95, LNP-Eg95-1, LNP-Eg95-2, and LNP-Eg95-3). The RNA to total liposome volume ratio was 3:1, and the flow rate ratio was 9 mL / min to 3 mL / min. After sample collection, an equal volume of citrate-sodium citrate solution (pH 4.0) was immediately added for dilution. The samples were then processed using Millipore microfluidic reagents. The 30 kDa ultrafiltration tube was centrifuged to 1 / 4 of its original volume, and then 15 times the volume of PBS buffer was added to reduce the ethanol content to below 0.5%. Finally, ultrafiltration was performed using PBS buffer containing 10% sucrose to concentrate the mRNA to a concentration of 50 μg / ml. The mRNA vaccine was obtained by aseptic filtration.

[0061] The average particle size of LNP particles was measured using dynamic light scattering on a Malvern Zetasizer Nano-ZEN 3600 (Malvern) potential-laser particle size analyzer. The particle size range was 90-120 nm, the polydispersity index (PDI) was less than 0.3, and the surface potential was negative. The encapsulation efficiency was determined to be no less than 90% using the Quant-iT™ RiboGreen™ RNA Assay Kit (Invitrogen™ R11490). The physical parameters of the samples met the requirements (results are shown in Table 4).

[0062] Table 4 Characterization of LNP-mRNA samples

[0063]

[0064] Further analysis was conducted to determine the expression levels of Eg95 protein in mammalian cells using four circular RNA vaccines (LNP-Eg95, LNP-Eg95-1, LNP-Eg95-2, and LNP-Eg95-3). HEK-293 cells were cultured at 5 × 10⁻⁶ cells / year. 4Cells were seeded per well in cell culture plates. When the cell confluence reached 30-50%, the cell culture medium was aspirated, and 0.5 μg of the circular RNA vaccine was added per well for transfection. Untransfected cells served as a blank control. Twenty-four hours after transfection, Eg95 protein expression in each group was analyzed by Western blot. HRP-mediated mouse anti-echinococcosis Eg95 monoclonal antibody (Shanghai Kebiao Biotechnology Co., Ltd., catalog number KB41752) was used. Results are as follows... Figure 3 As shown, no signal was detected at the target protein location in the blank control group, while signals were detected at the target protein location in the LNP-Eg95 group, LNP-Eg95-1 group, LNP-Eg95-2 group, and LNP-Eg95-3 group. This indicates that the four circular RNA vaccines prepared successfully expressed the Eg95 protein, the main antigenic protein of Echinococcus larvae, in mammalian cells.

[0065] Example 5: Sheep Immunization Test

[0066] Four-month-old healthy lambs were randomly divided into 9 groups of 5 lambs each. Groups 1, 2, 3, and 4 were injected with LNP-Eg95, LNP-Eg95-1, LNP-Eg95-2, and LNP-Eg95-3 vaccines, respectively, at a dose of 1 ml per lamb, injected into the leg muscle. Group 5 was injected with a genetically engineered subunit vaccine for echinococcosis (hydatid disease) (Chongqing Aolong Biological Products Co., Ltd.), at a dose of 1 ml per lamb, injected subcutaneously in the neck; this was the commercial subunit vaccine group. Group 6 was the untreated control group (blank control group). Group 7 was the LNP-Eg95-a vaccine control group. The preparation methods were as described in Examples 1-4. The mutant of the Eg95 protein constructed in Example 1 (refer to patent CN108066755B) was a truncated EG95 amino acid sequence with 8 amino acids shortened at the N-terminus, 24 amino acids shortened at the C-terminus, and amino acid ET inserted at positions 62 and 63 (amino acid sequence as shown in SEQ ID). As shown in NO.21, the two mutated EG95 protein sequences were linked together using a flexible linker (amino acid sequence as shown in SEQ ID NO.22). A universal signal peptide (amino acid sequence as shown in SEQ ID NO.23) was added to the N-terminus to aid protein expression and secretion, resulting in the Eg95-a protein (amino acid sequence as shown in SEQ ID NO.24). The codon-optimized Eg95-a nucleotide sequence is shown in SEQ ID NO.25. Group 8 was the LNP-Eg95-b vaccine control group. The preparation method was as described in Examples 1-4. In Example 1, the mutant Eg95 protein was constructed by truncating 8 amino acids at the N-terminus of the EG95 amino acid sequence, truncating 24 amino acids at the C-terminus, and inserting amino acid ET (amino acid sequence as shown in SEQ ID NO.21) at positions 62 and 63. An immune-enhancing sequence 2 was linked to the C-terminus via linker arm 1, and a universal signal peptide (amino acid sequence as shown in SEQ ID NO.23) was added to the N-terminus to aid protein expression and secretion, resulting in the Eg95-b protein (amino acid sequence as shown in SEQ ID NO.24). As shown in NO.26), the codon-optimized Eg95-b nucleotide sequence is shown in SEQ ID NO.27; Group 9 is the LNP-Eg95-c vaccine control group, and the preparation method is as described in Examples 1-4. In Example 1, the immune enhancement sequence (refer to patent CN117467018A) uses immune enhancement sequence 3 (i.e., a mutant of the heavy chain constant region fragment of sheep IgG, whose amino acid sequence is shown in SEQ ID NO.28), with a signal peptide (amino acid sequence shown in SEQ ID NO.29) added at the N-terminus to help protein expression and secretion. The EG95 antigen and the sheep antibody Fc protein fragment are linked through a linker rich in glycine and serine (amino acid sequence shown in SEQ ID NO.30) to obtain the Eg95-c protein amino acid sequence as shown in SEQ ID NO.As shown in Figure 31, the codon-optimized Eg95-c nucleotide sequence is shown in SEQ ID NO. 32.

[0067] Animal reactions were observed, and blood samples were collected before immunization and at 30, 60, 90 and 180 days after a single immunization to prepare serum samples. The antibodies against the main protective antigen Eg95 protein of Echinococcus hydatids were detected in the serum using the Echinococcus hydatids ELISA antibody detection kit (Chongqing Aolong Biological Products Co., Ltd.). The operation method is as described in the ELISA kit instructions. The result judgment criteria are as follows ([1] Wang Shufang, He Jingui. Detection of the immunization effect of the genetically engineered subunit vaccine for Echinococcus hydatid disease [J]. Chinese Journal of Veterinary Medicine, 2019(3):2.). If the sample OD450≤0.3, it is judged as negative for Echinococcus hydatids antibody, and the sample has no protective effect against Echinococcus hydatids infection; if the sample OD450>0.3, it is judged as positive for antibody. Samples with an OD450 between 0.3 and 0.6 are considered to have low protective efficacy, but still offer some protection against Echinococcus granulosus infection; samples with an OD450 ≥ 0.6 are considered to have protective efficacy, with higher antibody levels, and can provide more than 90% resistance in challenge experiments.

[0068] The results are shown in Tables 5 and 6. Before immunization, none of the groups had protective efficacy; 30 days after immunization, 5 / 5 of the immunization groups showed protective efficacy; 90 days after immunization, the protective efficacy was observed in 1 / 5, 3 / 5, 5 / 5, 5 / 5, 3 / 5, 3 / 5, 5 / 5, and 1 / 5 of the LNP-Eg95 group, LNP-Eg95-1 group, LNP-Eg95-2 group, LNP-Eg95-3 group, LNP-Eg95-a group, LNP-Eg95-b group, LNP-Eg95-c group, and the commercial subunit vaccine group, respectively; 180 days after immunization, the protective efficacy was observed in the LNP-Eg95 group, LNP-Eg95-1 group, LNP-Eg95-2 group, LNP-Eg95-3 group, LNP-Eg95-a group, LNP-Eg95-b group, and LNP-Eg95-c group. The Eg95-c group and the commercial subunit vaccine group showed protective efficacy at 0 / 5, 1 / 5, 3 / 5, 5 / 5, 1 / 5, 1 / 5, 3 / 5, and 0 / 5 doses, respectively. The LNP-Eg95-3 group required only one dose to provide 180 days of immune protection (protection rate of over 70%); while the commercial subunit vaccine produced significant antibodies 30 days after one dose, and then the antibody levels gradually decreased, with no protective efficacy after 180 days.

[0069] Table 5. Serum anti-Eg95 antibody (OD450 value) in 4-month-old lambs after a single immunization with vaccines group 1-4

[0070] ((+) indicates protective ability; no marking indicates low or no protective ability)

[0071]

[0072] Table 6. Serum anti-Eg95 antibody (OD450 value) in 4-month-old lambs after a single immunization with Group 5-9 vaccine.

[0073] ((+) indicates protective ability; no marking indicates low or no protective ability)

[0074]

[0075] Example 6: Mouse Immunization Test

[0076] Eight-week-old female BALB / c mice were randomly divided into three groups of five each. Group 1 received LNP-Eg95-3 vaccine at a dose of 1 / 10 (100 μL / mouse) injected into the leg muscle. Group 2 received a genetically engineered subunit vaccine against echinococcosis (hydatid disease) from Chongqing Aolong Biological Products Co., Ltd. at a dose of 1 / 10 (100 μL / mouse) injected subcutaneously on the back; this was the commercial subunit vaccine group. Group 3 was the control group, which did not receive any injections. Animal responses were observed.

[0077] Bone marrow was collected 30 days post-immunization for Eg95 protein-specific long-lived plasma cells. The methods were as follows (doi: 10.1038 / s41541-023-00638-6): Bone marrow was collected from the femur and tibia of mice using a syringe and injected into FACS buffer. The cells were filtered through a 63 μm Nitex mesh filter, and erythrocytes were lysed on ice with ACK solution for 5 minutes. The bone marrow cells were then resuspended in pre-chilled culture medium at 1×10⁻⁶ cells / mL. 6Bone marrow cells were first treated with Fc receptor blocking reagent and live / dead cell double staining reagent (Invitrogen / ThermoFisher Scientific, 1:1000) for 30 min, washed twice with FACS buffer, and stained with plasma cell population-specific fluorescently labeled antibody at 4°C for 30 min. After incubation, the cells were washed twice with FACS buffer and fixed / permeabilized under light conditions using eBioscience intracellular fixation and permeabilization buffer set (ThermoFisher Scientific) at 4°C for 40 min. Recombinant Echinococcus granulosus Eg95 protein (Shanghai Kebiao Biotechnology Co., Ltd., catalog number KB11057) was prepared to obtain Eg95-FITC and Eg95-Alexa Fluor647 using either the FITC conjugation kit (Abcam) or the Alexa Fluor 647 conjugation kit (Abcam). Cells were stained with Eg95-FITC and Eg95-Alexa Fluor 647 at 4°C for 30 min; then washed twice with permeabilization buffer and resuspended in FACS buffer. Data were analyzed using a BD LSR II flow cytometer, acquired using Diva software (BD Bioscience), and analyzed using FlowJo software (BD Bioscience).

[0078] See results Figure 4 The proportions of Eg95 protein-specific long-lived plasma cells (CD3-CD45R-CD138+) in the bone marrow of mice in the control group, the commercial subunit vaccine group, and the LNP-Eg95-3 group were 0%, 0%, and 1.46%, respectively. Antigen-specific long-lived plasma cells were almost undetectable in the control group and the commercial vaccine group, while the LNP-Eg95-3 vaccine successfully induced the production of Eg95 protein-specific long-lived plasma cells. This constitutes one of the important mechanisms by which the LNP-Eg95-3 vaccine achieves immune protection with a single injection.

[0079] Example 7: Preparation and Immunological Activity Analysis of Linear mRNA and Self-Replicating mRNA Vaccines Expressing Eg95-3 Protein

[0080] (1) Construction and preparation of linear mRNA molecules expressing Eg95-3 protein

[0081] The template gene for constructing the linear mRNA molecule (nucleotide sequence shown in SEQ ID NO.19) contains, from the 5' end to the 3' end, the EcoRI restriction site sequence, the T7 promoter sequence, the 5' untranslated region (5' UTR), the Eg95-3 nucleotide sequence, the 3' untranslated region (3' UTR), the poly(A)tail, and the BsaI restriction site sequence. The template gene was inserted into the EcoRI / BsaI multiple cloning site of the pUC57 vector through homologous recombination to construct the template plasmid DNA, which was then transformed into E. coli DH5α, and sequencing confirmed its correctness. Following Example 2, *E. coli* fermentation and recombinant plasmid preparation were performed. Following Example 3, plasmid linearization and purification were conducted. A single-stranded linear mRNA was generated using the EasyCap T7 Co-transcription Kit with CAG Trimer (Novizan) as a template. This linear mRNA contained, from the 5' end to the 3' end, a Cap1 analog structure, a 5' UTR, the Eg95-3 nucleotide sequence, a 3' UTR, and a Poly(A) tail, wherein the uracil was modified with N1-methyl-pseudouracil. The mRNA transcript was purified by magnetic beads to remove impurities, yielding a linear mRNA molecule expressing the Eg95-3 protein. The purity was above 90% by agarose gel electrophoresis, meeting the requirements. This linear mRNA molecule was designated Eg95-3L (nucleotide sequence is shown in SEQ ID NO. 9).

[0082] (2) Construction and preparation of self-replicating mRNA molecules expressing Eg95-3 protein

[0083] The template gene for constructing the self-replicating mRNA molecule (nucleotide sequence shown in SEQ ID NO.20) contains, from the 5' end to the 3' end, the NdeI restriction site sequence, linker arm sequence 1, Eg95-3 nucleotide sequence, linker arm sequence 2, and ShpI restriction site sequence. The template gene was inserted into the NdeI and ShpI restriction sites of the T7-VEE-GFP vector (Addgene, #58977) through homologous recombination to construct the template plasmid DNA, which was then transformed into E. coli DH5α and sequenced to verify its correctness. Following Example 2, *E. coli* fermentation and recombinant plasmid preparation were performed. The plasmid was linearized using the MluI restriction endonuclease (nearshore protein), and purified using a DNA purification kit. A single-stranded self-replicating mRNA was generated using the T7 HighYield RNA Synthesis Kit for Co-transcription (Yisheng Biotechnology (Shanghai) Co., Ltd.) as a template. This self-replicating mRNA contained a Cap1 analog structure, a 5' UTR, a Venezuelan equine encephalitis (VEE) non-structural protein, a subgenomic promoter sequence (SGP), an Eg95-3 nucleotide sequence, a 3' UTR, and a Poly(A) tail, with uracil modified by N1-methyl-pseudouracil. The mRNA transcript was purified using lithium chloride according to the method in Example 3, and the purity of the self-replicating mRNA expressing the Eg95-3 protein was above 90% by agarose gel electrophoresis, meeting the requirements. This self-replicating mRNA molecule was designated Eg95-3SAM (nucleotide sequence shown in SEQ ID NO. 10).

[0084] (3) Preparation and analysis of linear mRNA vaccines and self-replicating mRNA vaccines expressing Eg95-3 protein

[0085] Linear mRNA nanoliposomes and self-replicating mRNA nanoliposomes were prepared using the LNP packaging kit (Navitech, catalog number: N01001050). The mRNA (numbered Eg95-3L and Eg95-3SAM, respectively) was diluted with citrate-sodium citrate solution (pH 4.0) (buffer phase). The concentration of the diluted mRNA was 100 μg / mL. The diluted mRNA and liposomes were mixed in a certain ratio using an LNP microfluidic preparation instrument (NexSTARnano1) to prepare LNPs (numbered LNP-Eg95-3L and LNP-Eg95-3SAM, respectively). The volume ratio of mRNA to total liposomes was 3:1, and the flow rate ratio was 9 ml / min to 3 ml / min. After the samples were collected, an equal volume of citrate-sodium citrate solution (pH 4.0) was added for dilution. The samples were centrifuged using a Millipore 30 kDa ultrafiltration tube to 1 / 4 of the original volume. Then, 15 times the volume of PBS buffer was added to reduce the ethanol content to below 0.5%. Finally, ultrafiltration was performed using PBS buffer containing 10% sucrose to concentrate the mRNA to a concentration of 50 μg / mL.

[0086] The average particle size of LNP-Eg95-3L was measured using dynamic light scattering on a Malvern Zetasizer Nano-ZEN 3600 (Malvern) with a polydispersity index (PDI) of 86 nm, a negative surface potential, and a surface potential of 0.13. The encapsulation efficiency was determined to be 91% using the Quant-iT™ RiboGreen™ RNA Assay Kit (Invitrogen™ R11490). The average particle size of LNP-Eg95-3SAM was 121 nm, with a PDI of 0.15 and a negative surface potential, and an encapsulation efficiency of 92%. The physical parameters of both samples met the requirements (results are shown in Table 7).

[0087] Table 7 Characterization of LNP-mRNA samples

[0088]

[0089] (4) In vitro expression verification

[0090] The expression activities of LNP-Eg95-3L and LNP-Eg95-3SAM in HEK-293 cells were analyzed according to Example 4. The results are as follows: Figure 5 As shown, no signal was detected at the target protein location in the blank control group, while signals were detected at the target protein location in both the LNP-Eg95-3L group and the LNP-Eg95-3SAM group, indicating that both RNA vaccines successfully expressed the Eg95 protein, the main antigenic protein of Echinococcus larvae, in mammalian cells.

[0091] (5) Immunization test on sheep

[0092] Four-month-old healthy lambs were randomly divided into three groups of five lambs each. Groups 1 and 2 were injected with LNP-Eg95-3L and LNP-Eg95-3SAM, respectively, at a dose of 1 ml per lamb, injected into the leg muscle. Group 3 was the control group, which did not receive any injections. Animal responses were observed. Blood samples were collected before immunization and at 30, 90, and 180 days after the first immunization, and 60 serum samples were prepared.

[0093] Referring to the method in Example 5, the ELISA antibody detection kit for Echinococcus hydatids (hydatid larvae) (Chongqing Aolong Biological Products Co., Ltd.) was used to detect the antibodies against the Eg95 protein, the main protective antigen of Echinococcus hydatids (hydatid larvae), in serum.

[0094] The results are shown in Table 8. Before immunization, none of the groups provided protection; at 30 and 90 days after immunization, 5 / 5 of the immunization groups showed protection; at 180 days after immunization, 4 / 5 and 4 / 5 of the LNP-Eg95-3L and LNP-Eg95-3SAM groups, respectively, provided protection; while the blank control group showed no protection. The LNP-Eg95-3L and LNP-Eg95-3SAM groups required only one dose to provide 180 days of immune protection (protection rate over 70%), indicating that both linear mRNA vaccines and self-replicating mRNA vaccines against Eg95-3 have good immunogenicity, providing a foundation for further vaccine development.

[0095] Table 8. Serum anti-Eg95 antibody (OD450 value) in 4-month-old lambs after a single immunization with each group of vaccines.

[0096] ((+) indicates protective ability; no marking indicates low or no protective ability)

[0097]

[0098] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A circular RNA molecule encoding a modified Echinococcus granulosus Eg95 protein, characterized in that, The nucleotide sequence of the circular RNA molecule is SEQ ID NO.

8.

2. The circular RNA molecule according to claim 1, characterized in that, The modified Echinococcus granulosus Eg95 protein was obtained by linking an immunostimulating sequence to the C-terminus of the Echinococcus granulosus Eg95 protein mutant via a linker arm; The amino acid sequence of the Echinococcus granulosus Eg95 protein is shown in SEQ ID NO.14; the mutant of the Echinococcus granulosus Eg95 protein is the E at position 52 mutated to K, the E at position 93 mutated to K, and the P at position 123 mutated to Q. The sequence of the connecting arm is SEQ ID NO.15; the sequence of the immune enhancement is SEQ ID NO.

18.

3. The circular RNA molecule according to claim 2, characterized in that, The amino acid sequence of the modified Echinococcus granulosus Eg95 protein is SEQ ID NO.3; and / or, the nucleotide sequence encoding the modified Echinococcus granulosus Eg95 protein is SEQ ID NO.

7.

4. A linear mRNA encoding a modified Echinococcus granulosus Eg95 protein, characterized in that, The nucleotide sequence of the linear mRNA is shown in SEQ ID NO.

9.

5. A self-replicating mRNA encoding a modified Echinococcus granulosus Eg95 protein, characterized in that, The nucleotide sequence of the self-replicating mRNA is shown in SEQ ID NO.

10.

6. A genetically engineered vaccine, characterized in that, The genetically engineered vaccine is prepared by mixing a circular RNA molecule as described in any one of claims 1-3, a linear mRNA as described in claim 4, or a self-replicating mRNA as described in claim 5 with liposomes.

Images