A CagW mRNA-LNP composition and its application in the preparation of products for treating Helicobacter pylori infection.

Abstract

This invention relates to a Cag W mRNA-LNP composition and its application in the preparation of products for treating Helicobacter pylori infection. The composition comprises (i) mRNA encoding an open reading frame of Cag W protein or an immunogenic fragment thereof; and (ii) an LNP nanoparticle delivery carrier. The sequence of the mRNA encoding Cag W protein is shown in SEQ ID NO. 1 and 3, and the amino acid sequence is shown in SEQ ID NO. 2 and 4. Compared with the prior art, the Cag W mRNA-LNP vaccine prepared by this invention has high immunogenicity, targeting, potent humoral immunity, comprehensive and ideal cellular immunity, 100% challenge protection efficacy, and effective bacterial clearance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a Cag W mRNA-LNP composition and its application in the preparation of products for treating Helicobacter pylori infection. Background Technology

[0002] Helicobacter pylori ( Helicobacter pylori It is a Gram-negative bacterium. H. pylori Infections are closely associated with the development and exacerbation of various stomach diseases, including chronic gastritis and peptic ulcers, and are a leading risk factor for stomach cancer. The International Agency for Research on Cancer (IARC), a branch of the World Health Organization (WHO), has classified infection as a major risk factor for stomach cancer. H. pylori It is clearly classified as a Group 1 carcinogen.

[0003] at present, H. pylori The standard clinical treatment for infections is triple or quadruple therapy, the core components of which include a proton pump inhibitor (PPI) and two or three antibiotics (such as amoxicillin, clarithromycin, metronidazole, etc.). However, the effectiveness of this regimen is facing serious challenges. The increasing prevalence of antibiotic resistance worldwide has led to a significant decline in the eradication rate of standard therapies. For example, in many regions, the resistance rate to clarithromycin has soared to over 15%, forcing clinicians to turn to more complex, multi-drug, and more side-effect-prone bismuth quadruple therapy (BQT).

[0004] Therefore, in the face of the significant challenge of antibiotic resistance, there is an urgent need to develop a non-antibiotic intervention, especially an effective preventative vaccine, to control the problem at its source. H. pylori The spread and infection.

[0005] at present H. pylori Vaccine development is extremely challenging, including the latest mRNA-LNP platform technology, whose antigen selection is highly focused on a few key virulence factors: (1) CagA (cytotoxin-associated gene A protein): a major oncogenic protein, considered a key candidate antigen.

[0006] (2) VacA (vacuolating cytotoxin A): another major virulence factor that can induce cell vacuolization and apoptosis.

[0007] (3) UreB (urease B subunit): Urease is H. pylori UreB is a key enzyme essential for neutralizing stomach acid and surviving in highly acidic environments. It is the main immunogenic subunit of the enzyme.

[0008] Although antigens such as CagA, VacA, and UreB have shown some potential in animal models, vaccines targeting these antigens have not yet demonstrated sufficiently stable and durable protective efficacy in human clinical trials. This suggests that targeting these traditional virulence factors alone may not be enough to induce a comprehensive and effective protective immune response. Summary of the Invention

[0009] The purpose of this invention is to overcome the existing... H. pylori To address shortcomings such as insufficient immune protection against vaccine targets, this paper provides a CagW mRNA-LNP composition and its application in the preparation of products for treating Helicobacter pylori infection.

[0010] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide a Cag W mRNA-LNP composition, comprising: (i) mRNA encoding the open reading frame (ORF) of the Cag W protein or its immunogenic fragment; (ii) LNPs nanodelivery carriers.

[0011] In some specific embodiments, the mRNA sequence encoding the Cag W protein is shown in SEQ ID NO.1, and the amino acid sequence of the Cag W protein is shown in SEQ ID NO.2.

[0012] SEQ ID NO.1: SEQ ID NO.2: MFNIKRTFLVTIISFFLIIPNWLKAIDLPIVSNLKIYQTVYCMLIPSYVLTNKSFADILTGYTSIGASGSGKSSGQGVIEALSTPLATSLAASNLVKYLNTLGPLWGSAWASVATAIQGFALTPSSGCNFGWN ALINKNIDVSMDSVLDNLSNKIQNFTKGGVEDNVKGNILLQIIGSITAQASTNITADGLIWLIGKEFTANKLQNNTTAMLAFAALESVVKGADAAVLPAYGVVNLPDIIIGQGSYLDFVSYLIYIVFGIFVFIS FMKLRDISNGIQLNIGFEYMRFVGGTLFKMAMVSFIAYAGFGYLYKISYSIYFGLAGAFGLNQVLFWALDLVLNYTVNSILPAVRAVFSNVGNNAPSLLQGLQVAGISLFAIFMQVTIIMRISTVVVKPLIAG AFSGIVFPIAVCLIVLDWFKDSMKNILIWFINNLFILVLAIPILLFGVLALLAFNLTITPSVAIQNINQGGLGIDSTIASLITLFILKGFIETIIESVNAIVNTIFSSVSMDGSRMDRERDALMVGRVGGSMFK In some specific embodiments, the mRNA is also an mRNA encoding a Cag W-Fc protein fused with an Fc fragment, wherein the Fc fragment is selected from human IgG1 Fc fragment and IgG Fc fragment.

[0013] In some specific embodiments, the mRNA sequence encoding the Cag W-Fc fusion protein is shown in SEQ ID NO.3, and the amino acid sequence of the Cag W-Fc fusion protein is shown in SEQ ID NO.4.

[0014] SEQ ID NO.3: SEQ ID NO.4: MFNIKRTFLVTIISFFLIIPNWLKAIDLPIVSNLKIYQTVYCMLIPSYVLTNKSFADILTGYTSIGASGSGKSSGQGVIEALSTPLATSLAASNLVKYLNTLGPLWGSAWASVATAIQGFALTPSSGCNFGWN ALINKNIDVSMDSVLDNLSNKIQNFTKGGVEDNVKGNILLQIIGSITAQASTNITADGLIWLIGKEFTANKLQNNTTAMLAFAALESVVKGADAAVLPAYGVVNLPDIIIGQGSYLDFVSYLIYIVFGIFVFIS FMKLRDISNGIQLNIGFEYMRFVGGTLFKMAMVSFIAYAGFGYLYKISYSIYFGLAGAFGLNQVLFWALDLVLNYTVNSILPAVRAVFSNVGNNAPSLLQGLQVAGISLFAIFMQVTIIMRISTVVVKPLIAGA FSGIVFPIAVCLIVLDWFKDSMKNILIWFINNLFILVLAIPILLFGVLALLAFNLTITPSVAIQNINQGGLGIDSTIASLITLFILKGFIETIIESVNAIVNTIFSSVSMDGSRMDRERDALMVGRVGGSMFKG In some specific embodiments, the LNPs nanodelivery carrier comprises ionizable lipids, auxiliary phospholipids, sterols, and polyethylene glycol-modified lipids. The ionizable lipid is selected from any one of Lipid-SP-001, Lipid-SP-002, SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA, wherein the structure of Lipid-SP-001 is as follows: ; The structure of Lipid-SP-002 is as follows: .

[0015] In some specific embodiments, the auxiliary phospholipid is selected from one or more of distearylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), 1,2-distearyl-sn-glycero-3-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine, and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine. The sterol is selected from one or more of cholesterol, β-sitosterol, cholesterol, cholesterol ketone, 7β-hydroxycholesterol, and 7α-hydroxycholesterol; The PEGylated lipid is selected from one or more of the following: distearylphosphatidyl-polyethylene glycol, 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol, 1,2-distearyl-rac-glycero-3-methoxy polyethylene glycol, 1,2-dipalmitoyl-rac-glycero-3-methoxy polyethylene glycol, and 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine-methoxy polyethylene glycol.

[0016] The second technical solution of the present invention is to provide a method for preparing the CagW mRNA-LNP composition as described in one of the above technical solutions, comprising the following steps: Ionizable lipids, cofactor phospholipids, sterols, and polyethylene glycol-modified lipids were dissolved in ethanol to form the lipid phase. Prepare an aqueous phase containing mRNA with an open reading frame encoding Cag W protein or its immunogenic fragment; The lipid and aqueous phases were mixed using a microfluidic system, and the resulting mixture was dialyzed to obtain the CagW mRNA-LNP composition.

[0017] In some specific embodiments, the molar ratio of the ionizable lipid, cofactor phospholipid, sterol, and polyethylene glycol-modified lipid is (12-18):(30-55):(20-45):(0.5-2.5). The lipid phase with a concentration of (7.5-20) mM was mixed with the aqueous phase with a concentration of 0.1 mg / mL at a volume ratio of 1:(2-3).

[0018] The third technical solution of the present invention is to provide the application of the Cag W mRNA-LNP composition described in one of the above technical solutions in the preparation of products for the prevention or treatment of Helicobacter pylori infection.

[0019] In some specific embodiments, the product is a vaccine for the prevention or treatment of Helicobacter pylori infection.

[0020] Compared with the prior art, the present invention has the following advantages: (1) High immunogenicity and targeting: The LNPs nanodelivery carrier of the present invention can effectively deliver Cag W mRNA and can be targeted to organs such as spleen and liver. In particular, it can be targeted to the spleen, an immune organ rich in antigen-presenting cells, and induces high immunogenicity in mice.

[0021] (2) Strong humoral immunity: The Cag W-Fc mRNA-LNP vaccine of the present invention can induce the production of high titers of IgG and IgM antibodies specific to Cag W protein.

[0022] (3) Comprehensive and ideal cellular immunity: The Cag W-Fc mRNA-LNP vaccine of the present invention induces strong cell-mediated immunity and significantly upregulates the secretion of multiple key cytokines, including IL-2, IFN-γ, IL-12 (Th1 response, which is essential for the clearance of intracellular pathogens), IL-4 (Th2 response, which helps B cells produce antibodies) and IL-17A (Th17 response, which is essential for the clearance of mucosal pathogens). This confirms that the Cag W-Fc mRNA-LNP vaccine induces clearance H. pylori The Th1 / Th2 / Th17 mixed immune response required for infection.

[0023] (4) 100% protection against viral attack: In H. pylori In the live bacteria challenge model, mice immunized with the Cag W-FcmRNA-LNP vaccine of this invention showed a 100% survival rate.

[0024] (5) Effective bacterial clearance: Compared with the control group, the bacterial load in the liver and stomach of immunized mice was effectively reduced. Attached Figure Description

[0025] Figure 1 Bioinformatics screening of the Cag W protein for this invention. (A) Solubility; (B) Secondary structure; (C) Tertiary structure; (D) Ramachandran plot analysis; (E) Z-score.

[0026] Figure 2 This is a structural element of the Cag W mRNA molecule of this invention.

[0027] Figure 3The physicochemical properties characterization diagram of Cag W-Fc mRNA-LNP of the present invention is shown. (A) Average hydrated particle size of LNP; (B) Polydispersity index (PDI); (C) Encapsulation efficiency of LNP-mRNA; (D) Zeta potential.

[0028] Figure 4 This is a schematic diagram of the in vivo targeted distribution of Cag W-Fc mRNA-LNP of the present invention.

[0029] Figure 5 This is a graph showing the ELISA results of the Cag W specific antibody titers (IgG and IgM) of this invention.

[0030] Figure 6 This is a Western Blot result of the specific detection of the antibody induced in mice by the present invention.

[0031] Figure 7 This is a graph showing the cytokine secretion levels after restimulation of spleen cells in mice immunized according to the present invention.

[0032] Figure 8 The diagram shows (A) of the mice challenged with the virus according to the present invention and (B) of the mice's survival curve.

[0033] Figure 9 This is a statistical graph showing the bacterial colony count (CFU) of the stomach and liver of mice immunized according to the present invention.

[0034] Figure 10 The images show H&E staining of various parts (stomach, heart, liver, spleen, lung, and kidney) in mice immunized according to the present invention (A); liver function (ALT, AST, ALB) and kidney function (CRE, UREA) indicators (B). Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0037] Unless otherwise specified, the materials and processes described in the following embodiments or examples are conventional materials and processes used in the art to achieve the corresponding functions.

[0038] Example 1: Screening, design, and preparation of target mRNA for Cag W antigen (1) This embodiment uses software tools and an online server (Protein-Sol server (https: / / protein-sol.manchester.ac.uk / )) to predict the solubility of Cag W protein, such as... Figure 1 As shown in A.

[0039] The secondary structure of the Cag W protein was predicted using the PSIPRED server (http: / / bioinf.cs.ucl.ac.uk / psipred / ), such as... Figure 1 As shown in B.

[0040] The tertiary structure of the Cag W protein was predicted using 3Dpro server (Scratch Protein Predictor (uci.edu)). Figure 1 As shown in C.

[0041] The conformational rationality of the Cag W protein was analyzed using the GalaxyRefine web server (https: / / galaxy.seoklab.org / cgi-bin / submit.cgi?type=REFINE). Generally, if the proportion of amino acid residues falling in the allowed and maximally allowed regions of the protein is higher than 90%, the conformation of the model can be considered to conform to the rules of stereochemistry. Figure 1 As shown in D, the proportion of amino acid residues in the Cag W protein that fall within the allowed and maximally allowed regions is higher than 90% of the total protein.

[0042] The Z-score of Cag W protein was analyzed using the ProSA web server, such as... Figure 1 As shown in E.

[0043] Based on the above comprehensive assessment, H. pylori The highly conserved membrane protein CagW is a candidate antigen for this invention.

[0044] (2) Construction of plasmid pIVTRup-Cag W-Fc (2-1) The ORF sequence encoding Cag W and the sequence encoding human IgG1 Fc fragment are codon optimized during gene synthesis by using a flexible linker sequence to obtain the optimized Cag W-Fc fusion gene sequence (e.g., SEQ ID NO.5).

[0045] SEQ ID NO.5: (2-2) The Cag W-Fc fusion gene sequence (SEQ ID NO.5) was cloned into the pIVTRup vector to construct the recombinant vector pIVTRup-Cag W-Fc expressing the Cag W-Fc fusion gene.

[0046] The constructed recombinant vector pIVTRup-Cag W-Fc was transformed into... E. coli TOP10, select positive clones for identification and culture, and achieve large-scale amplification of recombinant plasmid pIVTRup-Cag W-Fc.

[0047] (3) In vitro transcription to synthesize the target mRNA (3-1) DNA template preparation: The DNA of the recombinant plasmid pIVTRup-Cag W-Fc was linearized using XbaI enzyme as a template, incubated at 37°C for 90 min, and purified using an enzyme digestion product recovery kit.

[0048] (3-2) In vitro transcription DNA templates were transcribed in vitro using a mixture of NTPs in the presence of T7 RNA polymerase according to the NEB HiScribe® T7 High Yield RNA Synthesis Kit (catalog number: E2040S). After the reaction, the template was digested with DNase I, and mRNA quality was assessed using Nanodrop.

[0049] Using an in vitro transcription kit, an anti-reverse capanalog (ARCA) was co-transcribed with T7 RNA polymerase to ensure its correct orientation and formation of a cap structure on the mRNA. The 5'UTR, 3'UTR, and Poly(A) tail were derived from the pIVTRup plasmid. Using the linearized recombinant plasmid as a template, a fully formed and mature Cag W-Fc mRNA (structure shown in Figure 1) was finally synthesized. Figure 2 (As shown).

[0050] Example 2 Preparation of mRNA-LNP 1. Preparation of the lipid phase: Ionizable lipid (Lipid-SP-001), 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 15:45:38.5:1.5, with a total lipid concentration of 12.5 mM.

[0051] The structure of the ionizable lipid (Lipid-SP-001) is as follows: 2. Preparation of aqueous phase: The Cag W-Fc mRNA prepared in Example 1 (or the mRNA synthesized by the biotechnology company as described in SEQ ID NO. 3) was dissolved in 50 mM citrate buffer at pH 4.0, with a concentration of 0.1 mg / mL.

[0052] 3. Mixing: Using a microfluidic mixing system (Precision NanoSystems' Ignite), the lipid phase and aqueous phase were mixed at a flow rate of 1:3 and a total flow rate of 12 mL / min to obtain the Cag W-Fc mRNA-LNP stock solution.

[0053] 4. Purification: The collected LNP stock solution was immediately diluted with PBS at pH 7.4 and dialyzed and concentrated using a tangential flow filtration (TFF) system (Sartorius, Hydrosart® ultrafiltration membrane, 100 kDa) to remove ethanol and replace the buffer, resulting in a concentrated suspension of Cag W-Fc mRNA-LNP.

[0054] 5. Sterile filtration: The Cag W-Fc mRNA-LNP concentrated suspension was sterilized by passing it through a 0.22 μm filter membrane and stored at -80°C.

[0055] like Figure 3 The figure shows the physicochemical properties of Cag W-Fc mRNA-LNP. (A) shows the average hydrated particle size of LNP, approximately 98 nm; (B) shows the polydispersity index (PDI) of Cag W-Fc mRNA-LNP is 0.12; (C) shows the encapsulation efficiency of LNP-CagW-Fc mRNA is greater than 95%; (D) shows the Zeta potential of Cag W-Fc mRNA-LNP is -1.2 mV (pH 7.4).

[0056] Example 3: In vivo biodistribution of Cag W-Fc mRNA-LNP The Cag W-Fc mRNA in Example 1 was replaced with an equimolar amount of Cy5-labeled Cag W-Fc mRNA, and Cy5-mRNA-LNP (Group 1 of this invention, targeting the spleen) and Cy5-mRNA-LNP (Group 2 of this invention, using standard liver-targeting lipid SM-102 instead of Lipid-SP-001) were prepared according to the method in Example 2.

[0057] Two groups of mRNA-LNP were administered to BALB / c mice via tail vein injection (iv) (6-8 weeks, 18-20 g, female, 1 μg (based on mRNA) / mouse). Mice were sacrificed 24 h after injection, and major organs (lymph node, spleen, lung, liver, heart, and kidney) were collected.

[0058] The fluorescence intensity of isolated organs was detected and quantitatively analyzed using an IVIS in vivo imaging system, such as... Figure 4 As shown, the fluorescence signal of Group 1 (LNP-Cy5-mRNA) in the spleen was significantly higher than that of Group 2 (LNP-Cy5-Mrna (SM102)) in the liver, and the fluorescence signal was the strongest. It can be seen that both Group 1 and Group 2 have liver targeting. In addition, Group 1 also has spleen targeting.

[0059] Example 4 Animal Immunization The experimental animals used were 6-8 week old female C57BL / 6 mice, weighing 18-20 g, and were divided into the following groups: ① PBS control group supplemented with alum aluminum adjuvant (concentration 0.5 μg / μL, denoted as alum); ② empty LNP group (containing no mRNA, labeled as LNP or mock); ③ Cag W-Fc mRNA-LNP group (Group 1 of this invention, labeled as Cag W mRNA-LNP); ④ Cag W-Fc fusion protein group (amino acids as shown in SEQ ID NO.4, labeled as CagW protein); 10 mice in each group.

[0060] Mice were fed normally and immunized three times on days 0, 14 and 28, with each injection containing 1 μg (based on mRNA).

[0061] (1) Humoral immunity Serum was collected via orbital blood sampling on days 14, 28, and 42. The titers of CagW-specific IgM, IgG, and their subtypes IgG1 and IgG2a antibodies in the serum were detected using the coated CagW-Fc fusion protein. ELISA results are shown below. Figure 5 As shown in the figure. The pooled serum from day 35 was used as the primary antibody to detect its specific binding to the Cag W-Fc fusion protein. Western blot results are shown in the figure. Figure 6 As shown.

[0062] Figure 5 and Figure 6The results showed that only the Cag W-Fc mRNA-LNP group induced high titers of specific anti-Cag W antibodies IgM, IgG and their subtypes IgG1 and IgG2a.

[0063] (2) Cellular immunity After euthanizing the mice, spleen cells were collected and processed at 10... 5 Cells were seeded in 96-well plates and restimulated in vitro with 10 μg / mL Cag W-Fc fusion protein / LNP-Cag W-Fc mRNA / empty LNP. After 72 hours of culture, cell supernatant was collected. The concentrations of cytokines (IL-2, IL-4, IL-12p70, IFN-γ, IL-17A) were detected using a CBA (Cytometric Bead Array) or an ELISA kit (all ELISA kits were purchased from Yaxin Biotechnology).

[0064] like Figure 7 As shown, spleen cells in the Cag W-Fc mRNA-LNP group significantly secreted IL-2, IL-4, IL-12, IFN-γ, and IL-17A after restimulation.

[0065] This result confirms a mixed response of Th1 cells (secreting IFN-γ, IL-12, IL-2), Th2 cells (secreting IL-4), and Th17 cells (secreting IL-17A), which is crucial for simultaneously clearing intracellular and extracellular (mucosal) pathogens. H. pylori It is of utmost importance.

[0066] Example 5: Animal challenge protection like Figure 8 As shown in Figure A, mice were subjected to [immunization] on day 42 after the start of immunization. H. pylori (ATCC 43504) Live bacteria challenge, oral gavage 4×10 8 CFU / each, once a day, for a total of 4 days.

[0067] Continuously observe the mice's condition, weight changes, and survival, such as Figure 8 As shown in B, all mice in the PBS group and the empty LNP group died (or showed severe symptoms) within 4 days, while the survival rate of the Cag W-Fc mRNA-LNP group was 100%.

[0068] After the challenge experiment (day 63), all surviving mice were euthanized, and stomach and liver tissues were aseptically harvested. The stomach and liver tissues were homogenized and serially diluted, plated, and incubated for 72 hours before colony counting. Figure 9The quantitative comparison results of bacterial colony forming units (CFU) showed that the bacterial load in the stomach and liver of the Cag W-Fc mRNA-LNP group was significantly lower than that of the control group, confirming its strong bacterial clearance ability.

[0069] Example 6: Security Assessment During animal immunization and challenge, the weight changes and general health status of all mice were monitored regularly. Twenty-four hours post-immunization, histopathological examination (H&E staining) was performed on various sites (stomach, heart, liver, spleen, lung, and kidney) to assess local inflammatory responses, such as… Figure 10 As shown in Figure A, the Cag W-FcmRNA-LNP group and the Cag W-Fc fusion protein group showed no obvious tissue damage, while the empty LNP group and the PBS group showed severe tissue damage and significant cell infiltration.

[0070] On day 35 post-immunization, serum samples were collected to measure liver function (ALT, AST, ALB) and kidney function (CRE, UREA) indicators. Figure 10 As shown, there were no significant differences in serum biochemical indicators and histopathological examination between the Cag W-Fc mRNA-LNP group mice and the PBS control group.

[0071] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A CagW mRNA-LNP composition, characterized in that, include: (i) mRNA encoding an open reading frame of the Cag W protein or an immunogenic fragment thereof; (ii) LNPs nanodelivery carriers.

2. The CagW mRNA-LNP composition according to claim 1, characterized in that, The mRNA sequence encoding the Cag W protein is shown in SEQ ID NO.1, and the amino acid sequence of the Cag W protein is shown in SEQ ID NO.

2.

3. The CagW mRNA-LNP composition according to claim 1, characterized in that, The mRNA is also an mRNA encoding a Cag W-Fc protein fused with an Fc fragment, wherein the Fc fragment is selected from human IgG Fc and IgG1 Fc fragments.

4. The CagW mRNA-LNP composition according to claim 1, characterized in that, The mRNA sequence encoding the Cag W-Fc fusion protein is shown in SEQ ID NO.3, and the amino acid sequence of the Cag W-Fc fusion protein is shown in SEQ ID NO.

4.

5. The CagW mRNA-LNP composition according to claim 1, characterized in that, The LNPs nanodelivery carrier comprises ionizable lipids, auxiliary phospholipids, sterols, and polyethylene glycol-modified lipids. The ionizable lipid is selected from any one of Lipid-SP-001, Lipid-SP-002, SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA, wherein the structure of Lipid-SP-001 is as follows: ； The structure of Lipid-SP-002 is as follows: 。 6. The CagW mRNA-LNP composition according to claim 5, characterized in that, The auxiliary phospholipid is selected from one or more of the following: distearylphosphatidylcholine, dioleoylphosphatidylethanolamine, 1,2-distearyl-sn-glycero-3-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine, and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine. The sterol is selected from one or more of cholesterol, β-sitosterol, cholesterol, cholesterol ketone, 7β-hydroxycholesterol, and 7α-hydroxycholesterol; The PEGylated lipid is selected from one or more of the following: distearylphosphatidyl-polyethylene glycol, 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol, 1,2-distearyl-rac-glycero-3-methoxy polyethylene glycol, 1,2-dipalmitoyl-rac-glycero-3-methoxy polyethylene glycol, and 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine-methoxy polyethylene glycol.

7. A method for preparing the CagW mRNA-LNP composition according to any one of claims 1 to 6, characterized in that, Includes the following steps: Ionizable lipids, cofactor phospholipids, sterols, and polyethylene glycol-modified lipids were dissolved in ethanol to form the lipid phase. Prepare an aqueous phase containing mRNA with an open reading frame encoding Cag W protein or its immunogenic fragment; The lipid and aqueous phases were mixed using a microfluidic system, and the resulting mixture was dialyzed to obtain the CagW mRNA-LNP composition.

8. The preparation method according to claim 7, characterized in that, The molar ratio of the ionizable lipids, cofactor phospholipids, sterols, and PEGylated lipids is (12-18):(30-55):(20-45):(0.5-2.5). The lipid phase with a concentration of (7.5-20) mM was mixed with the aqueous phase with a concentration of 0.1 mg / mL at a volume ratio of 1:(2-3).

9. The use of the CagW mRNA-LNP composition as described in any one of claims 1 to 6 in the preparation of products for the prevention or treatment of Helicobacter pylori infection.

10. The application according to claim 9, wherein the product is a vaccine for the prevention or treatment of Helicobacter pylori infection.

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