An Omp25 mRNA-LNP composition and use thereof in the manufacture of a medicament for treating brucella infection

Abstract

The present application relates to an Omp25 mRNA-LNP composition and its application in the preparation of a product for treating brucella infection diseases, the composition comprising (i) mRNA encoding an antigen targeting Omp25 protein; (ii) LNP nano delivery carrier. The mRNA sequence encoding the antigen targeting Omp25 protein is shown as SEQ ID NO. 1, and the translated amino acid sequence is shown as SEQ ID NO. 2; the mRNA sequence encoding the antigen targeting Omp25 protein is shown as SEQ ID NO. 3, and the translated amino acid sequence is shown as SEQ ID NO. 4. Compared with the prior art, the Omp25 mRNA-LNP vaccine prepared by the present application has the effects of high immunogenicity, targeting, strong humoral immunity, comprehensive and ideal cellular immunity, 100% attack protection efficacy and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an Omp25 mRNA-LNP composition and its application in the preparation of products for treating Brucella infections. Background Technology

[0002] Brucella ( Brucella bacillus Brucella is a type of Gram-negative intracellular parasite, belonging to the zoonotic pathogens. It is mainly transmitted to humans and animals through contact with the secretions of infected animals (such as cattle, sheep, and pigs) or by consuming undercooked, contaminated animal products. Brucella infection poses significant threats to human health, manifesting as: acute infection with high fever (undulant fever), chills, joint pain, and hepatosplenomegaly; if left untreated, it can easily become chronic, leading to long-term fatigue, nerve damage, cardiovascular complications, and even lifelong disability. In animals, Brucella infection can cause abortion in cows, orchitis in bulls, and infertility in sheep and goats, severely restricting the development of animal husbandry and causing huge economic losses.

[0003] Currently, the clinical treatment of Brucella infection mainly relies on combination antibiotics, such as doxycycline + rifampin. However, this approach has significant drawbacks: the treatment period is as long as 6-8 weeks, the incidence of side effects (such as liver damage and gastrointestinal reactions) is high, and the relapse rate for patients with chronic infection exceeds 15%. Furthermore, since antibiotics cannot prevent infection, there is an urgent need to develop safe and effective preventive vaccines to address the transmission risks brought about by large-scale livestock farming and global population movement.

[0004] Despite decades of research into Brucella vaccines, existing vaccines still face significant bottlenecks, and an ideal vaccine that simultaneously meets the criteria of "high protection rate, high safety, and broad-spectrum coverage" has yet to emerge. Among these, animal vaccines pose the following safety risks: Currently, veterinary clinical practice mainly uses live attenuated vaccines (such as S19 vaccine for cattle and Rev.1 vaccine for sheep). These vaccines carry the risk of "reversion to ancestral virus" (which may lead to disease in animals) and are teratogenic to pregnant animals (e.g., S19 vaccine can cause abortion in cows). Furthermore, they cannot distinguish between "natural infection" and "vaccine immunization" (DIVA principle), which hinders the eradication of the disease.

[0005] Therefore, in the face of major challenges such as antibiotic issues and safety concerns with existing Brucella vaccines, there is an urgent need to develop a new Brucella vaccine that can simultaneously meet the requirements of "high protection rate, high safety, and broad-spectrum coverage". Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing Brucella vaccines, such as low safety, and to provide an Omp25 mRNA-LNP composition and its application in the preparation of products for treating Brucella infections.

[0007] 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 an antigen targeting the Omp25 protein, wherein the antigen is a protein with amino acids as shown in SEQ ID NO.2, or a recombinant fusion protein linked to an Fc fragment, wherein the Fc fragment is selected from any one of human IgG Fc, IgG1 Fc, and IgG2 Fc.

[0008] In some specific embodiments, the amino acid sequence of the recombinant fusion protein linking the Fc fragment is shown in SEQ ID NO. 4.

[0009] The second technical solution of the present invention is to provide an Omp25 mRNA-LNP composition, comprising: (i) mRNA encoding an antigen targeting the Omp25 protein as described in one of the above technical solutions; (ii) LNPs nanodelivery carriers.

[0010] In some specific embodiments, the sequence of the mRNA encoding the antigen targeting the Omp25 protein is shown in SEQ ID NO.1, and its translated amino acid sequence is shown in SEQ ID NO.2.

[0011] SEQ ID NO.1: AUGAACGGAAGAGUGGAUUAUUUGGUCACUGAGGAAGAGAUCAAUCUUACCAAGAGGCCCUCAGGGCUGGGCUUCAACAUCGUCGGUGGGACAGAUCAGCAGUAUGUCUCCAACGACAGUGGCAUCUACGUCAGCCGCAUCAAAGAAAAUGGGGCUGCGGCCCUGGAUGGGCGGCUCCAGGAGGGUGAUAAGAUCCUUUCGGUAAAUGGCCAAGACCUA AAGAACCUGCUGCACCGGAAUGCUGUAGACCCUCUUUCGUAAUGCAGGCUAUGCUGUGUCUCUGAGAGUGCAGCACAGGUUACAGGUGCAGAAUGGACCUAUAGGACAUCGAGGUGAAGGGGACCCGGAUGGUAUUCCCAUAUUUAUGGUGCUGGUGCCAGUGUUUGCCCUCACCAUGGUAGCAGCCUCCCCUUUCAUGAGAUACCGGCAACAACUUUGA SEQ ID NO.2: MASNGLYRVALAASPYLEUVALTGLUGLUEILEASNLTHRLYSRPROSERGLEUGLYFASNILEVGLYGLYTASPGLNQTYRVALSASNASPSGLYILEYVALSERRIlELYSEASNGLYAALAALASPGLYRLEUGLNEGLYASPKILELEUSVALASNGGLNASP LLYSASNLLEUHISRASNALAVASPLEUFARGASNAGLYTYRAVALSERLARGVALQHISARGLGLNVALQASNGLYPILEGLYHARRGGLYEGLYASPPASPGLYIPROILEFMETVALLVALPROVPHEALALTHRMETVALAALASPROPHEMARGTYRRGLNGLNL The mRNA sequence encoding the antigen targeting the Omp25-Fc protein is shown in SEQ ID NO.3, and its translated amino acid sequence is shown in SEQ ID NO.4.

[0012] SEQ ID NO.3: SEQ ID NO.4: IASNTHRTHISTYRRASPALAMLYSARGGLEUCYSCVALLEULLEUCYSGALAVALFVALSERAARGMETNGLYARGVASPTYRLVALTHREGLUGLUIASNLEUTLYSARGPSERGLYLGLYPHENILEVALGGLYTHRDGLNGLNYVALSERNASPSERGILETYRVSERARGILYSGLUNGLYALAAALALEUDGLYARG LGLNGLUGASPLYSILEUSERVASNGLYQASPLEUKASNLEULHISARGNALAVALDLEUPHERASNALAGTYRALAVSERLEURVALGLNHARGLEUQVALGLNNGLYPROIGLYHISRGLYGLUGASPPRODGLYILEPILEPHEMVALLEUVPROVALFALALEUTMETVALAALASERPPHEMETRTYRARGQGLNLEU 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-Bru-001, SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA, wherein the structure of Lipid-SP-Bru-001 is as follows: .

[0013] 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, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, and sphingomyelin. The sterol is selected from one or more of cholesterol, β-sitosterol, cholesterol, cholesterol ketone, cholesterol, 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.

[0014] The third technical solution of the present invention is to provide a method for preparing the Omp25 mRNA-LNP composition as described in the second technical solution above, 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 encoding an antigen targeting the Omp25 protein; The lipid and aqueous phases were mixed using a microfluidic system, and the Omp25 mRNA-LNP composition was obtained after dialysis.

[0015] In some specific embodiments, the molar ratio of the ionizable lipid, cofactor phospholipid, sterol, and polyethylene glycol-modified lipid is (10-19):(30-55):(20-45):(0.5-3). 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).

[0016] The fourth technical solution of the present invention is to provide the application of the Omp25 mRNA-LNP composition as described in the second technical solution above in the preparation of products for the prevention or treatment of Brucella infection.

[0017] In some specific embodiments, the product is a vaccine for the prevention or treatment of Brucella infection.

[0018] Compared with the prior art, the present invention has the following advantages: (1) High efficiency targeting: This invention constructs an Omp25 mRNA-LNP system to achieve high efficiency targeting of tissues such as the liver and spleen. In particular, after intravenous injection of LNP composed of Lipid-SP-Bru-001, the accumulation of LNP in the spleen is significantly higher than that of the standard liver-targeting LNP (SM-102), and the fluorescence intensity ratio of spleen to liver reaches 6.8:1, which lays the foundation for cellular immune response; (2) Broad-spectrum protection: Omp25 mRNA-LNP has a protective effect on mice immunized with Brucella 16M challenge; (3) Strong cellular immune response: Induces a th1-type cellular immune response. After immunized mouse spleen cells are restimulated by Omp25 recombinant protein, the IFN-γ secretion level reaches 926±85 pg / mL (significantly higher than the 412 pg / mL of the control group), effectively clearing intracellular bacteria (the core requirement for Brucella infection); the IL-4 secretion level reaches 139±16 pg / mL (significantly higher than the 50 pg / mL of the control group).

[0019] (4) Durable humoral immune response: Induces high titers of Omp25-specific IgG antibodies, with the antibody titer (GMT) reaching 1.5 × 10⁻⁶ after three immunizations. ^ 7. Furthermore, the IgG2a / IgG1 ratio is 3.2:1 (a characteristic of Th1-type immune response). This antibody can mediate ADCC effects, kill infected cells, and prevent bacterial spread.

[0020] (5) 100% protection efficacy against challenge: In the Brucella 16M strain lethal dose challenge model, the survival rate of mice immunized with the vaccine of this invention reached 100%, and there were no chronic infection symptoms (such as hepatosplenomegaly), while the survival rate of PBS group and LNP group was 0.

[0021] (6) Excellent safety: No risk of atavism: The mRNA does not integrate into the genome, and Omp25 has no toxic fragments, avoiding the pathogenic risk of live attenuated vaccines; No organ toxicity: Serum ALT and AST levels were normal in immunized mice, and no inflammatory infiltration was observed in spleen and liver tissue pathology. Attached Figure Description

[0022] Figure 1 Figure A shows the physicochemical characterization of the Omp25-Fc mRNA-LNP of this invention. Figure B shows the LNP particle size distribution curve determined by dynamic light scattering (DLS); Figure C shows the transmission electron microscopy (TEM) image.

[0023] Figure 2 This is an in vivo targeting (biodistribution) experiment diagram of Omp25-Fc mRNA-LNP of the present invention. Fluorescence imaging of major organs (spleen, liver, lung, kidney, and heart) in mice 24 hours after intravenous injection of Luc mRNA-LNP and standard SM102-LNP.

[0024] Figure 3 This is a graph showing the ELISA detection results of the Omp25-Fc mRNA-LNP specific antibody titers (IgG, IgG1, IgG2a) of this invention.

[0025] Figure 4 This is a graph showing the cellular immune response detection results of Omp25-Fc mRNA-LNP in this invention (Th1 cytokines and CTL killing rate).

[0026] Figure 5 To assess the survival rate of immunized animals after challenge with Brucella bacteria.

[0027] Figure 6 Microscopic images of tissue sections from immunized animals.

[0028] Figure 7 Statistical graphs of liver function (ALT, AST) and kidney function indicators (CRE, UREA) in immunized animals.

[0029] Figure 8 This is a Western blotting pattern showing the specific binding of immunized animal serum to the Omp25-Fc fusion protein. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Example 1: Preparation of mRNA from an antigen targeting the Omp25 protein (1) Construction of plasmid pIVTRup-Omp25-Fc (1-1) The candidate antigen fragment targeting the Omp25 protein was tandemly linked with the sequence encoding the human IgG1 Fc fragment through a flexible linker sequence. Codon optimization was performed during gene synthesis (using GenSmart codon optimization software to remove rare codons (such as AGG, CGG) and potential secondary structures (regions with ΔG < -30 kcal / mol) to obtain the optimized Omp25-Fc fusion gene sequence, the coding strand of which is shown in SEQ ID NO.5).

[0034] SEQ ID NO.5: (1-2) The Omp25-Fc fusion gene sequence (such as SEQ ID NO.5) was cloned into the pIVTRup vector to construct the recombinant vector pIVTRup-Omp25-Fc expressing the Omp25-Fc fusion gene.

[0035] The constructed recombinant vector pIVTRup-Omp25-Fc was transformed into... E. coli TOP10, select positive clones for identification and culture, and achieve large-scale amplification of recombinant plasmid pIVTRup-Omp25-Fc.

[0036] (2) In vitro transcription to synthesize the target mRNA (2-1) DNA template preparation: The DNA of recombinant plasmid pIVTRup-Omp25-Fc was linearized downstream of the 3' UTR using XbaI enzyme as a template, incubated at 37°C for 90 min, and purified using an enzyme digestion product recovery kit.

[0037] (2-2) 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.

[0038] Using an in vitro transcription kit, an anti-reverse cap analog (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 (SEQ ID NO.6), 3'UTR (SEQ ID NO.7), and Poly(A) tail were derived from the pIVTRup plasmid. Using the linearized recombinant plasmid as a template, a fully formed and mature Omp25-Fc-Fc-mRNA was finally synthesized.

[0039] SEQ ID NO.6 (5' UTR): GCCACCATGGTGAGCAAGGCCTGCTTCTCCCCTGCTGCTGGTGGTGGCGGCTGCTCCGGTG SEQ ID NO.7 (3' UTR): AGCTTGCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC Example 2: Preparation of mRNA-LNP 1. Preparation of lipid phase: Ionizable lipid (Lipid-SP-Bru-001), sphingomyelin, cholesterol and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000) were dissolved in anhydrous ethanol at a molar ratio of 10:50:38:2, and the total lipid concentration was 15 mM.

[0040] The structure of the ionizable lipid (Lipid-SP-Bru-001) is as follows: 2. Preparation of aqueous phase: The Omp25-Fc mRNA prepared in Example 1 (or the mRNA synthesized by the biotechnology company as described in SEQ ID NO. 3) was dissolved in 20 mM sodium acetate buffer (containing 0.01% EDTA) at pH 4.0, with a concentration of 0.1 mg / mL.

[0041] 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 20 mL / min. The mixing chamber temperature was 25℃ to obtain the Omp25-Fc mRNA-LNP stock solution.

[0042] 4. Purification: The collected LNP stock solution was immediately diluted 5-fold with PBS at pH 7.4, and buffer was replaced using a tangential flow filtration (TFF) system (Sartorius, Hydrosart® ultrafiltration membrane, 100 kDa): transmembrane pressure 15 psi, flow rate 10 mL / min, replacement volume 10 times column volume, until the ethanol content < 0.5%; then concentrated to a final mRNA concentration of 1 mg / mL Omp25-Fc mRNA-LNP concentrated suspension.

[0043] 5. Sterile filtration: The Omp25-Fc mRNA-LNP concentrated suspension was sterilized by passing it through a 0.22 μm filter membrane and stored at -80°C.

[0044] The physicochemical properties of the prepared Omp25-Fc mRNA-LNP were characterized as follows: (1) Particle size and PDI: Omp25-Fc mRNA-LNP suspension was diluted to 100 μg / mL with PBS containing 5% sucrose and measured at 25℃ using Zetasizer Nano ZS90 (Malvern). Each sample was tested 3 times and the average value was taken.

[0045] The results are as follows Figure 1 As shown in A, the average hydrated particle size is 75 ± 5 nm, and the PDI is 0.085 ± 0.02.

[0046] (2) Zeta potential: Under the same dilution conditions as above, the Zeta potential was measured using a Zetasizer Nano ZS90. The Zeta potential at pH 7.4 was -2.5 ± 1.2 mV (close to neutral, reducing non-specific adsorption and complement activation).

[0047] (3) Encapsulation efficiency (EE%): Two groups were set up using the RiboGreen RNA Quantitative Reagent Kit (Thermo Fisher): Total mRNA group: Take 10 μL of Omp25-Fc mRNA-LNP suspension, add 1 μL of 1% Triton X-100 to permeate the membrane, and incubate for 5 min; Free mRNA group: Take 10 μL of Omp25-Fc mRNA-LNP suspension and add RiboGreen reagent directly; Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm, and calculated using the formula: EE% = (total mRNA fluorescence intensity - free mRNA fluorescence intensity) / total mRNA fluorescence intensity × 100%.

[0048] The encapsulation efficiency was calculated to be 96.5 ± 1.8%, and the high encapsulation efficiency can reduce the degradation of free mRNA.

[0049] (4) Transmission electron microscopy (TEM) observation: Take 10 μL of Omp25-Fc mRNA-LNP suspension, add it to a copper grid, stain with 2% phosphotungstic acid for 5 min, and observe with Tecnai G2 F20 TEM (FEI).

[0050] The results are as follows Figure 1 As shown in B, Omp25-Fc mRNA-LNP is spherical, uniformly dispersed, and without aggregation, with a particle size consistent with that measured by DLS.

[0051] Example 3: In vivo biodistribution of Omp25-Fc mRNA-LNP In Example 2, the Omp25-Fc mRNA was replaced with an equimolar amount of Luc-labeled Omp25-Fc mRNA, and Luc-mRNA-LNP (Group 1 of this invention, targeting the spleen) and Luc-mRNA-LNP (Group 2 of this invention, using standard liver-targeting lipid SM-102 instead of Lipid-SP-Bru-001) were prepared according to the method of Example 2.

[0052] Two groups of mRNA-LNP were administered via tail vein injection (iv) to C57BL / 6 mice (6-8 weeks, 18-22 g, female, 10 μg (based on mRNA) / mouse). Mice were sacrificed 24 h after injection, and major organs (lymph nodes, spleen, lungs, liver, heart, and kidneys) were collected.

[0053] The fluorescence intensity of isolated organs was detected and quantitatively analyzed using an IVIS in vivo imaging system, such as... Figure 2 As shown, the fluorescence signal of Group 1 (Lipid-SP-Bru-001-LNP) in the spleen was significantly higher than that of Group 2 (SM102-LNP), and both groups showed fluorescence signals in the liver. This indicates that both Group 1 and Group 2 have liver targeting specificity. Furthermore, Group 1 also exhibits spleen targeting specificity; the fluorescence intensity of Group 1 in the spleen was 6.8 times that of Group 2, and the fluorescence intensity in the liver was 1 / 5 that of Group 2, resulting in a spleen / liver ratio of 6.8:1 (compared to 0.2:1 for Group 2).

[0054] Example 4 Animal Immunization The experimental animals used were 6-8 week old female C57BL / 6 mice, weighing 18-22 g, and were divided into the following groups: ① PBS control group supplemented with aluminum alum adjuvant (concentration 0.5 μg / μL, denoted as PBS); ② empty LNP group (containing no mRNA, labeled as LNP); ③ Omp25-Fc mRNA-LNP group (the group of this invention, labeled as Lipid-SP-Bru-001-LNP); ④ Omp25 protein group (labeled as Omp25 Protein), with 10 mice in each group.

[0055] Mice were fed normally and immunized three times on days 0, 14 and 28, with 1 μg (based on mRNA) injected via the tail vein each time, and the injection volume was 200 μL / mouse.

[0056] (1) Humoral immunity Serum was collected via orbital blood sampling on days 21 (7 days after the second immunization), 35 (7 days after the third immunization), and 42. The antibody titers of Omp25-specific IgG and its subtypes IgG1 and IgG2a in the serum were detected using the coated Omp25 fusion protein. ELISA results are shown below. Figure 3 As shown in the figure. The pooled serum from day 35 was used as the primary antibody to detect its specific binding to the Omp25-Fc fusion protein. Western blot results are shown below. Figure 8 As shown.

[0057] Figure 3 , 8 The results showed that only the Omp25-Fc mRNA-LNP group induced high-titer, specific anti-Omp25 antibody IgG and its subtypes IgG1 and IgG2a.

[0058] (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 Omp25-Fc fusion protein. After 72 hours of culture, cell supernatant was collected. The concentrations of cytokines (IL-4, IFN-γ) were detected using a CBA (Cytometric Bead Array) or an ELISA kit (all ELISA kits were purchased from Yageo Biotechnology).

[0059] like Figure 4 As shown, spleen cells in the Omp25-Fc mRNA-LNP group significantly secreted IL-4 and IFN-γ after restimulation.

[0060] Example 5: Animal challenge protection On day 42 after the start of immunization, mice were challenged with live Brucella 16M strain (ATCC 23456) via oral gavage, using 500 μL of bacterial suspension (containing 5 × 10⁻⁶ bacteria). 5 CFU / each, once a day, for a total of 4 days.

[0061] Continuously observe the mice's condition, weight changes, and survival, such as Figure 5 As shown, the survival rate of mice in the present invention group reached 100%, and their body weight recovered to the pre-infection level 15 days after challenge; the survival rate of the PBS group was 0%, and the survival rate of the LNP group was 0%.

[0062] Example 6: Security Assessment (1) During animal immunization and challenge, monitor the rate of change in body weight (rate of change in body weight = (body weight on the day - initial body weight) / initial body weight × 100%) and clinical symptoms (mental state, diet, activity level) of all mice regularly.

[0063] The rate of change in body weight of mice in this invention group was not significantly different from that in the PBS group (P>0.05), both remaining at +5% to +10%, with no adverse reactions such as lethargy, diarrhea, or fever.

[0064] (2) On day 35 post-immunization, serum samples were collected to detect liver function (ALT, AST) and kidney function (CRE, UREA) indicators. Figure 7 As shown, there were no significant differences in serum biochemical indicators and histopathological examinations between the Omp25-Fc mRNA-LNP group mice and the PBS control group.

[0065] The ALT=40±5 U / L, AST=130±8 U / L, CRE=20±5 μmol / L, and UREA=6.0±0.8 mmol / L in the Omp25-Fc mRNA-LNPs group were not significantly different from those in the PBS group (ALT=38±4 U / L, AST=135±7 U / L, CRE=21±4 μmol / L, and UREA=6.5±0.7 mmol / L) (P>0.05), and all were within the normal physiological range.

[0066] (3) Histopathological examination Mice were sacrificed on day 35, and spleen, liver, lung, heart, and kidneys were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5 μm thick), stained with H&E, and observed under an optical microscope (Olympus). The results are as follows Figure 6 As shown: No inflammatory cell infiltration (such as lymphocyte and neutrophil aggregation), tissue necrosis or abnormal proliferation was observed in any tissue of the Omp25-Fc mRNA-LNPs group, which was consistent with the tissue morphology of the PBS group, confirming that the vaccine has no local irritant effect.

[0067] 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. An antigen targeting the Omp25 protein, characterized in that, The antigen is a protein with an amino acid sequence as shown in SEQ ID NO.2, or a recombinant fusion protein linked to an Fc fragment, wherein the Fc fragment is selected from any one of human IgG Fc, IgG1 Fc, and IgG2 Fc.

2. The antigen targeting the Omp25 protein according to claim 1, characterized in that, The amino acid sequence of the recombinant fusion protein linking the Fc fragment is shown in SEQ ID NO.

4.

3. An Omp25 mRNA-LNP composition, characterized in that, include: (i) mRNA encoding an antigen targeting the Omp25 protein as described in claim 1 or 2; (ii) LNPs nanodelivery carriers.

4. The Omp25 mRNA-LNP composition according to claim 3, characterized in that, The mRNA sequence encoding the antigen targeting the Omp25 protein is shown in SEQ ID NO.1, and its translated amino acid sequence is shown in SEQ ID NO.2; The mRNA sequence encoding the antigen targeting the Omp25 protein is shown in SEQ ID NO.3, and its translated amino acid sequence is shown in SEQ ID NO.

4.

5. The Omp25 mRNA-LNP composition according to claim 3, 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-Bru-001, SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA, wherein the structure of Lipid-SP-Bru-001 is as follows: 。 6. The Omp25 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, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, and sphingomyelin. The sterol is selected from one or more of cholesterol, β-sitosterol, cholesterol, cholesterol ketone, cholesterol, 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 Omp25 mRNA-LNP composition as described in claim 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 encoding an antigen targeting the Omp25 protein; The lipid and aqueous phases were mixed using a microfluidic system, and the Omp25 mRNA-LNP composition was obtained after dialysis.

8. The preparation method according to claim 7, characterized in that, The molar ratio of the ionizable lipids, cofactor phospholipids, sterols, and polyethylene glycol-modified lipids is (10-19):(30-55):(20-45):(0.5-3). 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 Omp25 mRNA-LNP composition as described in claim 6 in the preparation of products for the prevention or treatment of Brucella infections.

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

Images