A method for preparing a vesicle for delivering ssie antigen, a nanovaccine and application thereof

Abstract

The present application belongs to the technical field of biology, and particularly relates to a vesicle preparation method for delivering SsIE antigen, a nano vaccine and application thereof. The present application finds that SsIE antigen is an important virulence factor and a potential vaccine candidate antigen of ETEC through screening, and can enhance adhesion and colonization ability in the process of colonization, so that the SsIE antigen can be prepared into a drug, especially a bacterial outer vesicle nano vaccine. The bacterial outer vesicle of the present application has no toxic side effects on cells, and can stimulate immune cells to induce strong antigen-specific immune response, and can be used for preventing and treating Escherichia coli infection and related diseases caused by the infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically to a method for preparing vesicles that deliver SsIE antigens, nanovaccines, and their applications. Background Technology

[0002] Diarrheal diseases are a major cause of high morbidity and mortality worldwide. Enterotoxigenic Escherichia coli (ETEC) is the most common bacterial pathogen, causing severe watery diarrhea, dehydration, and electrolyte imbalances in newborn animals such as piglets, calves, and lambs. It can also be transmitted through contaminated food and water, posing a serious threat to the health of children in developing countries with poor sanitation. Furthermore, it is the leading cause of "traveler's diarrhea" for international travelers to high-risk areas, indicating its typical zoonotic characteristics. Therefore, ETEC has become a significant pathogen threatening both livestock farming and public health. To date, clinical interventions for ETEC infection have primarily relied on oral rehydration and antibiotics. However, oral rehydration only alleviates the main symptoms and cannot eliminate the pathogen or block infection. Long-term, large-scale use of antibiotics can lead to the development of drug-resistant strains and the elimination of beneficial gut bacteria. Therefore, finding alternative treatments is crucial. Vaccines are an important means of preventing the serious consequences of ETEC infection. Whole-cell inactivated vaccines are a traditional strategy in ETEC vaccine development. Their protective efficacy highly depends on the complete preservation of the antigen's natural conformation. Compared to formaldehyde inactivation, studies have found that using polyurethane and targeted nucleic acid UVA photochemical inactivation (PUVA) can better preserve the natural conformation and function of ETEC surface antigens and virulence proteins, inducing broader and stronger specific IgG and anti-LT responses in mouse models; however, this method is more expensive. In contrast, live attenuated vaccines aim to stimulate a more comprehensive and durable mucosal immune response by mimicking the natural infection process, including immune responses against multiple ETEC colonization factors. These vaccines typically use genetic engineering techniques to remove or inactivate key toxin genes (such as LT and STa genes) or introduce safe mutations to construct strains with reduced virulence but preserved immunogenicity; however, their overall safety and stability still face some challenges. Utilizing novel antigen delivery platforms is an effective strategy to enhance the immune response of subunit vaccines, especially mucosal immunity. For example, developing nanovaccines using virus-like particles (VLPs) or bacterial outer membrane vesicles (OMVs) has become a new strategy in vaccine development. Summary of the Invention

[0003] The purpose of this invention is to develop a novel nanovaccine based on outer membrane vesicle delivery of SsIE antigen, contributing to the prevention and control of ETEC infection and the resulting human and animal diseases.

[0004] To achieve the objectives of this invention, the technical solution is as follows: In one aspect, the present invention discloses a method for preparing vesicles for delivering SsIE antigen, the method comprising: (1) The sequence containing the SsIE antigen is ligated to the linearized plasmid to obtain the recombinant plasmid; (2) Transform the recombinant plasmid into bacteria and screen for positive transformants; (3) The positive transformants were induced to express SsIE antigen and release outer membrane vesicles containing SsIE antigen. (4) Collect and purify the outer membrane vesicles containing SsIE antigen to obtain the product.

[0005] In one aspect, the present invention discloses a nanovaccine comprising vesicles capable of delivering SsIE antigens.

[0006] In some embodiments, the SsIE antigen sequence is as shown in SEQ ID NO.5.

[0007] In some embodiments, the vesicles delivering the SsIE antigen are bacterial extravesicles.

[0008] In one aspect, this invention discloses a bacterial outer membrane vesicle whose outer surface contains SsIE antigen. That is, the bacterial outer membrane vesicle described in this invention is capable of delivering SsIE antigen.

[0009] In some embodiments, the SsIE antigen is localized to the vesicle surface using a signal peptide and a transmembrane anchoring sequence.

[0010] In one embodiment, the signal peptide is derived from the Lpp protein, and the transmembrane anchoring sequence is derived from the OmpA protein.

[0011] In some embodiments, the bacterial outer membrane vesicles of the present invention contain a fusion protein Lpp-OmpA-SsIE located on the surface. This system enables the SsIE antigen bound to the vesicle to be displayed on the outer surface of the vesicle, wherein LPP provides a signal guiding peptide for the fusion protein and OmpA provides a transmembrane anchoring sequence for the fusion protein.

[0012] In one aspect, the present invention discloses a nucleotide sequence encoding the fusion protein Lpp-OmpA-SsIE, the sequence being shown in SEQ ID NO.1.

[0013] In one aspect, the present invention provides a fusion protein Lpp-OmpA-SsIE, the amino acid sequence of which is shown in SEQ ID NO.2.

[0014] In some embodiments, the nanovaccine of the present invention comprises bacterial exovesicles containing the fusion protein Lpp-OmpA-SsIE on their surface.

[0015] In one aspect, the present invention provides a vector plasmid containing the nucleotide sequence of the fusion protein Lpp-OmpA-SsIE as shown in SEQ ID NO. 1.

[0016] In one aspect, the present invention discloses a recombinant bacterium containing the aforementioned vector plasmid.

[0017] In one aspect, the present invention discloses a composition comprising the aforementioned bacterial outer membrane vesicles, fusion proteins, nucleotide sequences, vector plasmids, or recombinant bacteria.

[0018] In one aspect, this invention discloses the use of the aforementioned bacterial outer membrane vesicles, fusion proteins, nucleotide sequences, vector plasmids, or recombinant bacteria in the preparation of a drug. Preferably, the drug is a vaccine.

[0019] The drug / nano-vaccine of the present invention can be used to prevent / treat Escherichia coli infection. Preferably, the Escherichia coli is selected from one or more of enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), and enteroinvasive Escherichia coli (EIEC).

[0020] The drug / nanovice of the present invention can be used to prevent / treat diseases caused by Escherichia coli infection, including diarrheal diseases caused by Escherichia coli infection.

[0021] Beneficial effects This invention, through screening, discovered that the SsIE antigen is an important virulence factor and a potential vaccine candidate antigen for ETEC. It enhances adhesion and colonization during the colonization process and can be prepared into a drug, especially into a bacterial exovesicle nanovaccine.

[0022] The bacterial exovesicle nanovaccine for delivering SsIE antigen prepared in this invention has many advantages, including system stability (vesicles, antigens, and complexes are all stable); ease of storage and transportation; ability to stimulate a strong innate immune response (OMVs themselves are natural adjuvants); efficient delivery of SsIE to antigen-presenting cells and effective presentation; ability to induce a strong antigen-specific immune response; and ability to generate immune memory and provide long-term protection. It has good potential for widespread application.

[0023] The drug / nanovaccines of this invention can be used to prevent and treat Escherichia coli infection and related diseases. Attached Figure Description

[0024] Figure 1 PCR identification of Lpp-OmpA-ssIE (A) and SDS-PAGE detection of Lpp-OmpA-SsIE protein (B).

[0025] Figure 2 Size of SsIE fusion protein of coarsely separated OMVs (A), transmission electron microscopy observation (B), and Western blot detection of marker protein OmpF (C).

[0026] Figure 3 Results of OMVs-SsIE protease digestion (A), size of SsIE protein in purified OMVs (B), transmission electron microscopy observation (C), and NTA analysis (D).

[0027] Figure 4 Effects of different concentrations of OMVs-SsIE on HT-29 cells.

[0028] Figure 5 Gross anatomy (A), fecal characteristics score (B), survival rate (C), and weight change (D) of mice after being attacked with different concentrations of ETEC.

[0029] Figure 6 (A) Schematic diagram of the immunization procedure; (B) Measurement of IgG levels in each immunization group; (C) Measurement of IgG2a levels in each immunization group; (D) Measurement of IgG1 levels in each immunization group; (E) Measurement of IgA levels in each immunization group;* P <0.05; **, P <0.01; ****, P <0.0001.

[0030] Figure 7 Bacterial load in the intestines of mice after infection with ETEC (A), EPEC (B), and EIEC (C).

[0031] Figure 8 Histopathological observation (A) and pathological score (B) of mice immunized with ETEC infection.

[0032] Figure 9 Animal survival rate (A) and weight change (B) in protective experiments. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0034] Example 1: Construction, purification, and validation of engineered bacterial outer membrane vesicles (OMVs-SsIE) containing SsIE antigen 1.1 Construction of a fusion gene expression vector for displaying SsIE antigen Using the genomic DNA of wild-type enterotoxigenic Escherichia coli (ETEC) as a template, three pairs of specific primers were designed and synthesized to amplify... lpp, OmpA, ssIE Sequences. Primers Lpp-F and Lpp-R were used to amplify the gene fragment encoding the signal peptide (Lpp) (SEQ ID NO.3); primers OmpA-F and OmpA-R were used to amplify the gene fragment encoding the transmembrane anchoring sequence of outer membrane protein A (OmpA) (SEQ ID NO.4); primers ssIE-F and ssIE-R were used to amplify the gene fragment encoding the SsIE protective antigen (SEQ ID NO.5).

[0035] Table 1: Primer sequence list for gene PCR amplification ; Three target fragments were ligated using overlap PCR (SEQ ID NO.1). The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 5 min, 35 cycles; and 72℃ final extension for 5 min. After PCR, the target band size was confirmed by agarose gel electrophoresis, and the fragment was recovered. Agarose gel electrophoresis showed that the fusion fragment size was approximately 5661 bp.

[0036] 1.2 Preparation of linearized expression vectors for cloning The empty vector pET-32a was extracted from Escherichia coli. The empty vector was linearized by double digestion with restriction endonucleases EcoRI and XhoI. Then, the fusion gene obtained in Section 1.1 was ligated to the vector to construct the recombinant expression plasmid.

[0037] 1.3 Introduction of recombinant plasmids into expression host bacteria and screening for positive clones The constructed recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells via heat shock or electroporation. This strain integrates the T7 RNA polymerase gene into its genome and is induced by IPTG. The transformed bacterial culture was plated on LB agar plates containing 100 μg / mL ampicillin (Amp) and incubated upside down at 37°C for 12-16 hours. After single colonies grew on the plates, several colonies were randomly selected. A portion was subjected to rapid colony PCR verification using specific primers targeting the fusion gene, followed by electrophoresis to check for the expected band size. The other portion was inoculated into liquid culture medium, and plasmid was extracted for DNA sequencing to ensure complete gene sequence accuracy, without frameshifts or mutations. The positive clone strain verified by sequencing was formally named the Lpp-OmpA-SsIE engineered strain.

[0038] 1.4 Induction and Preliminary Validation of Genetically Engineered Bacteria The Lpp-OmpA-SsIE engineered bacteria were cultured in LB broth with shaking. When the OD600 value reached approximately 0.6, indicating that the bacteria were in mid-logarithmic growth, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mmol / L. To promote the expression of soluble proteins and reduce inclusion body formation, the culture conditions were adjusted to a lower temperature (25°C) and a slower rotation speed (110 rpm), and expression was induced for another 24 hours. After induction, the bacterial culture was centrifuged at 4°C and 5000×g for 15 minutes, and the bacterial pellet was collected. The bacterial cells were gently washed three times with pre-cooled phosphate-buffered saline (PBS, pH 7.4) to remove culture medium components. The washed bacterial cells were resuspended in 5 mL of PBS, and a mixture of protease inhibitors was added to prevent protein degradation. Subsequently, the bacterial suspension was disrupted using an ultrasonic homogenizer under ice bath conditions. The disrupted mixture was centrifuged at 7000×g for 45 minutes at 4°C to separate the supernatant (soluble component) and the precipitate (insoluble component, possibly containing inclusion bodies and cell debris). Small amounts of the supernatant and precipitate samples were taken separately and mixed with 5× protein loading buffer, then boiled in a 100°C metal bath for 15 minutes to fully denature the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was then performed using appropriate concentrations of separating and stacking gels. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250, and the gel was observed after destaining. By comparing the protein bands before and after induction, and between the supernatant and the precipitate, the expression level, solubility, and expression location of the recombinant Lpp-OmpA-SsIE fusion protein (the estimated molecular weight can be calculated based on the amino acid sequence) were analyzed to confirm successful protein expression.

[0039] 1.5 Extraction and purification of outer membrane vesicles from genetically engineered bacteria (1) The Lpp-OmpA-SsIE engineered bacteria were reactivated from the culture medium. A single colony was picked and inoculated into 20 mL of LB medium and cultured overnight at 37°C. The next day, the activated bacterial solution was transferred at a 1:100 inoculation ratio to a shake flask or fermenter containing 1000 mL of LB medium containing Amp and cultured at 37°C and 200 rpm. When the OD600 value of the culture medium reached about 0.6 again, IPTG was added to a final concentration of 0.5 mmol / L for induction. The culture conditions were then switched to 25°C and 110 rpm and induced for 24 hours to promote bacterial expression of the fusion protein and natural release of outer membrane vesicles. After induction, all bacterial solutions were centrifuged at 4°C and 5000×g for 15 minutes, and the supernatant containing outer membrane vesicles was collected. To ensure the removal of all remaining intact bacteria and large cell debris, the supernatant was centrifuged again under the same conditions, and then vacuum filtered or positive pressure filtered using a polyethersulfone (PES) membrane with a pore size of 0.22 μm to obtain a clear, sterile filtrate.

[0040] (2) Using ultrafiltration centrifuge tubes with a molecular weight cutoff of 100 kDa, perform ultrafiltration at 4°C and 5000 rpm to concentrate the filtrate to approximately 35 mL. Transfer the concentrate to a suitable high-speed centrifuge tube and centrifuge at 4°C and a relative centrifugal force of 45,000 × g for 3 hours using a high-speed centrifuge. After centrifugation, carefully discard the supernatant. A layer of black or dark brown translucent precipitate can be observed adhering to the bottom or side wall of the centrifuge tube. This is the crude extract containing outer membrane vesicles (OMVs-SsIE) displaying the SsIE antigen. Gently resuspend the precipitate in 200 μL of sterile PBS and disperse it completely by repeated pipetting or brief vortexing to obtain the crude extract of OMVs-SsIE. Store at 4°C for a short period or proceed to the next purification step immediately.

[0041] (3) Density gradient centrifugation was used to perform high-purity fine purification of the crude outer membrane vesicles to remove impurities such as co-precipitated protein aggregates and lipoprotein complexes. The outer membrane vesicles obtained by density gradient centrifugation were resuspended in 200 μL of sterile PBS with high-purity OMVs-SsIE precipitate. After thorough dissolution, they were stored at 4℃ or used immediately for subsequent characterization.

[0042] 1.6 Characterization of OMVs-SsIE extravesicles (1) Verification of antigen display and vesicle integrity by Western blotting: An appropriate amount of purified OMVs-SsIE sample was subjected to SDS-PAGE electrophoresis, and the protein was then transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with TBST buffer containing 5% skim milk powder at room temperature for 1 hour. Subsequently, the membrane was divided into two parts or incubated in parallel using different lanes. One part of the membrane was incubated overnight at 4°C with a monoclonal primary antibody against mouse anti-His tag (diluted at 1:2500) to specifically detect the displayed SsIE-His fusion protein; the other part of the membrane was incubated overnight at 4°C with a polyclonal antibody against rabbit anti-outer membrane vesicle marker protein OmpF (diluted at 1:500) to confirm that the extracted particles were indeed intact outer membrane vesicles. After incubation, the membrane was incubated for 1 hour at room temperature with goat anti-mouse IgG (diluted at 1:10000) or goat anti-rabbit IgG (diluted at 1:10000). After washing again, the luminescence was detected using enhanced chemiluminescence (ECL).

[0043] (2) Observe the morphology and size of OMVs-SsIEs using transmission electron microscopy (TEM): Take 5-10 μL of purified sample and drop it onto a carbon film copper grid treated with glow discharge. Let it stand at room temperature for about 1 minute to allow the vesicles to adsorb. Carefully absorb the excess liquid from the edge with filter paper. Then, immediately drop a drop of 2% (w / v) uranium acetate negative staining solution onto the copper grid, stain for 1 minute, and then absorb the staining solution again with filter paper. Allow it to air dry at room temperature. Place the prepared copper grid into the sample holder of the transmission electron microscope and observe it under a suitable accelerating voltage (e.g., 80 kV). A large number of well-defined, spherical or occasionally cup-shaped closed vesicle structures should be observed in the electron microscope image. The diameter is usually between 20-200 nm, which is the typical morphology of outer membrane vesicles.

[0044] (3) Quantitative analysis and particle size distribution analysis of vesicles were performed using nanoparticle tracking analysis (NTA) technology: The purified OMVs-SsIE sample was diluted appropriately with sterile PBS (usually 100-1000 times) to bring the particle concentration into the optimal detection range of the NTA instrument (approximately 10). 7 -10 9 Particles / mL). The diluted sample is injected into the sample cell and detected using an NTA instrument (such as the Malvern NanoSight NS300) equipped with a laser light source and a high-speed camera. The software calculates the hydration dynamic diameter of each particle by tracking the Brownian motion trajectory of each particle in the field of view according to the Stokes-Einstein equation, and statistically derives the absolute value of particle concentration and the particle size distribution map of the entire population (such as D10, D50, D90 values), thus providing key data on the particle size uniformity and concentration of vaccine candidates.

[0045] (4) Proteinase K digestion experiments were performed to analyze the topological localization of SsIE antigen on vesicles. Five parallel test samples were set up in EP tubes: ① Test group: An equal volume of purified OMVs-SsIE (e.g., containing 10 μg of protein) was mixed with proteinase K solution to achieve a final concentration of 0.02 mg / mL, and then incubated in a 37°C water bath for 10, 20, and 30 minutes, respectively; ② Proteinase K control group: An equal volume of proteinase K solution (without OMVs) was incubated at 37°C for 30 minutes; ③ OMVs-SsIE control group: An equal volume of purified OMVs-SsIE (without proteinase K) was incubated at 37°C for 30 minutes. After all incubation reactions were completed, pre-cooled protease inhibitors (such as PMSF) or SDS-PAGE loading buffer were added immediately and the mixture was boiled to terminate protease activity. Subsequently, all samples were analyzed by Western blotting, and the SsIE protein band was detected using an anti-His tag antibody. Expected Results and Analysis: If the Lpp-OmpA display strategy is successful, the SsIE antigen should be primarily localized on the outer surface of vesicles. In this case, the SsIE protein band in the test group (①) will significantly weaken or even disappear with prolonged proteinase K digestion time, indicating that the exposed antigen has been degraded by the protease. The OMVs-SsIE control group (③) should show a clear SsIE band. If the SsIE antigen is incorrectly encapsulated within the vesicle lumen or embedded in the membrane and not exposed, the band in the test group may be insensitive to proteinase K digestion. This test provides important functional evidence for antigen surface display.

[0046] Results related to Example 1: See Figure 1 Figure A shows the PCR results of the lpp-OmpA-ssIE fusion gene identification. Agarose gel electrophoresis showed that the size of the lpp-OmpA-ssIE fusion gene fragment in Figure A is approximately 5661 bp. Figure 1 Figure B shows the SDS-PAGE results of the Lpp-OmpA-SsIE fusion protein, with a molecular weight of approximately 206.5 kDa.

[0047] See Figure 2 Further detection using Western blot confirmed the presence of the target SsIE fusion protein. Figure 2 (A) Through transmission electron microscopy, it was found that the components separated from the supernatant were mostly nearly spherical vesicle-like substances. Figure 2 The sample (B) conforms to the reported morphology of OMVs and can be used for subsequent testing. Furthermore, SsIE detection of the OMV marker protein OmpF revealed a clear target band in the crude OMV extract. Figure 2(C). The results showed that OMVs carrying the target protein were successfully isolated.

[0048] See Figure 3 OMVs were digested using proteinase K. Western blot results after proteinase K digestion showed that the OMVs-SsIE target band was detected only in the untreated group. Figure 3 (A) confirmed that the SsIE protein was not encapsulated by a lipid membrane and was located on the surface of OMVs. In density gradient centrifugation purification, Optiprep (iodixanol) was removed from each layer after purification, and then each layer was analyzed by Western blot. Figure 3 In the third layer (B), relatively pure OMVs were observed under transmission electron microscopy. Figure 3 In the middle (C), the particle size and concentration were measured by nanoparticle tracking analysis (NTA). The highest concentration of particles with a diameter of about 100 nm was found, reaching 8 × 10⁻⁶. 6 Particle count / mL ( Figure 3 (D).

[0049] Example 2: In vitro safety test of OMVs-SsIE exovesicles The biosafety of OMVs-SsIE as a vaccine vector was evaluated in vitro, with a focus on its cytotoxicity. Human colon cancer cells HT29 were selected as the model cell line, commonly used to assess the biocompatibility of immunostimulants. Healthy HT29 cells in logarithmic growth phase were trypsinized, resuspended in complete culture medium (e.g., DMEM + 10% FBS), counted using a cell counting chamber, and the cell suspension density was adjusted. Cells were counted at 100 μL per well (approximately containing 5 × 10⁻⁶ cells). 3 Up to 1×10 4(Number of cells) The cell suspension was seeded into the wells of a 96-well cell culture plate, and the plate was gently shaken to distribute the cells evenly. The culture plate was placed in a cell culture incubator at 37°C and 5% CO2 and cultured overnight until the cells adhered and grew to approximately 80% confluence. The next day, the old culture medium in each well was carefully aspirated. Experimental group setup: 100 μL of fresh complete culture medium containing different final concentrations of OMVs-SsIE (25 μg / mL, 50 μg / mL, 100 μg / mL, based on protein concentration determination) was added to the cells. A blank control group was also set up: only 100 μL of fresh complete culture medium (without OMVs) was added. Five replicates were set up for each concentration group and control group to ensure data reliability. The culture plate was returned to the incubator and cultured for another 24 hours. After culture, the culture medium containing OMVs in each well was carefully aspirated. To avoid interference from residual OMVs, 200 μL of pre-warmed sterile PBS was added to each well, gently shaken, and then aspirated. This washing was repeated twice. After washing, add 110 μL of fresh basal culture medium (serum-free) and CCK-8 reagent mixture (10 μL of CCK-8 reagent) to each well. Return the culture plate to the incubator and incubate in the dark for 30 minutes to 2 hours, observing color changes in the wells during this period. After incubation, measure the absorbance (OD450) of each well at 450 nm using a multi-mode microplate reader. Calculate cell viability using the formula provided in the CCK-8 kit instructions: Cell viability (%) = [(Experimental group OD450 - Blank well OD450) / (Control group OD450 - Blank well OD450)] × 100%. By comparing cell viability at different OMVs-SsIE concentrations, a preliminary assessment can be made as to whether the vaccine candidate has significant toxicity to HT29 cells within the tested concentration range.

[0050] Results: After co-culturing HT-29 cells with different concentrations (100 μg / mL, 50 μg / mL, 25 μg / mL), CCK-8 assay results showed that cell survival was significantly increased in the 100 μg / mL OMVs group, while no significant differences were found in the other dosage groups. Overall, this indicates that OMVs-SsIE not only has no cytotoxic effect on cells, but also has the potential to promote cell proliferation or metabolic activity. Figure 4 ).

[0051] Example 3: Immunoprotective effect of OMVs-SsIE mucosal vaccine First, a mouse ETEC infection model was established for vaccine evaluation to determine the lethal dose required for subsequent challenge protection experiments. Twelve healthy female BALB / c mice aged 5 weeks were selected and acclimatized for 5 days in a standard laboratory animal room with a temperature of 20-26°C, relative humidity of 40%-70%, and 12-hour light-dark cycles. One day before challenge, the wild-type ETEC strain was thawed from glycerol cryopreservations and inoculated into fresh LB liquid medium, cultured at 37°C with shaking until mid-logarithmic growth (OD600 value approximately 0.6). Subsequently, bacterial cells were collected by centrifugation at 5000×g for 15 minutes at 4°C, the supernatant was discarded, and the bacterial pellet was resuspended in sterile PBS. The bacterial concentration was precisely adjusted to 1×10⁻⁶ cells / mL through serial dilutions. 6 CFU / mL, 5×10 6 CFU / mL and 1×10 7 Three gradients of CFU / mL.

[0052] Next, a challenge test was conducted. Twelve mice were randomly divided into three groups of four: a low-dose group, a medium-dose group, and a high-dose group. Each mouse was inoculated intraperitoneally with 200 μL of the corresponding concentration of ETEC bacterial solution. The specific dosage correspondence was as follows: the low-dose group was inoculated with 2 × 10⁻⁶ μL of ETEC bacterial solution. 5 CFU / animal, medium-dose group vaccinated with 1×10 6 CFU / animal, high-dose group vaccinated 2×10 6 CFU / mouse. After challenge, mice were observed for 7 consecutive days, and their survival status, weight changes, and clinical symptoms were recorded at fixed times each day.

[0053] Simultaneously, 12 hours after challenge, the fecal characteristics of each mouse were closely observed and recorded, and quantitatively scored according to the scoring criteria in Table 2-11: 0 points represent normally formed feces; 1 point represents relatively soft but still formed feces; 2 points represent feces that are pasty or semi-formed; and 3 points represent feces that are completely watery and loose. This score was used to assess the severity of diarrhea caused by ETEC infection.

[0054] During the observation period, any mice that died were immediately dissected to observe macroscopic pathological changes in major organs. At the end of the observation period on day 7, all surviving mice were euthanized and subjected to gross necropsy.

[0055] Finally, based on mortality data over 7 days, the median lethal dose (LD50) of ETEC in this strain of mice was calculated using the Karber method. 50 The calculation formula is: lg LD 50 = X k - d(Σp i - 0.5). Where, X kd is the logarithm of the concentration in the highest challenge dose group, d is the difference between the logarithms of two adjacent dose groups, and p is the concentration in the highest challenge dose group. i The mortality rate for each group (number of dead animals / total number of animals in the group), Σp i This represents the sum of mortality rates for each group. After calculating the logarithm, taking the antilogarithm yields the LD50 of ETEC in BALB / c mice. 50 Value, unit is CFU / piece. This LD 50 This value will serve as a key basis for determining the attack dose in subsequent vaccine challenge protection trials.

[0056] In determining LD 50 Subsequently, a formal evaluation test of the vaccine's immunization and protective efficacy against challenge was conducted. Forty-eight five-week-old female BALB / c mice were selected and acclimatized for five days under the same standard conditions. The mice were then randomly divided into four groups of 12 mice each: a PBS negative control group, an empty outer membrane vesicle control group (OMVs-WT), an OMVs-SsIE vaccine group, and a recombinant protein plus adjuvant positive control group (6P-1-SsIE).

[0057] Next, the immunization schedule was implemented. When the mice reached 6 weeks of age (day 1 of immunization, D1), the first immunization was initiated. Both the OMVs-SsIE vaccine group and the OMVs-WT control group were immunized via nasal drops, with each mouse receiving 70 μg of the corresponding OMVs (dissolved in an appropriate amount of sterile PBS). The 6P-1-SsIE group was immunized via subcutaneous injection. Before immunization, 50 μg of recombinant SsIE protein was mixed with an equal volume of adjuvant (such as aluminum adjuvant or Freund's incomplete adjuvant) on ice and shaken for 30 minutes to form an emulsion. Then, 200 μL was injected subcutaneously into each mouse. The PBS control group received 200 μL of sterile PBS subcutaneously into each mouse. A first booster immunization was administered on day 15 (D15), and a second booster immunization was administered on day 29 (D29). The dosage and route of administration remained unchanged for all three immunizations.

[0058] On day 3 after each immunization (i.e., D4, D18, D32), orbital venous blood was collected from mice in all groups. The blood samples were allowed to stand at 37°C for 1 hour to allow for full coagulation, and then centrifuged at 3000×g for 15 minutes at 4°C. The supernatant serum was carefully aspirated, aliquoted, and stored in an ultra-low temperature freezer at -80°C for subsequent antibody level detection.

[0059] Complete the entire immunization program (see Figure 6 Two weeks after the last blood collection (A), three mice were randomly selected from each group for challenge experiments. All challenged mice were weighed and recorded before challenge. Pre-determined ETEC wild-type LD50 strains were used. 50 Or higher (e.g., 2×LD) 50Mice were challenged with a lethal dose of the bacterial solution via intraperitoneal injection, with each mouse receiving 200 μL of the bacterial solution. After challenge, mice were observed for 7 consecutive days, with daily records kept of their survival status, weight changes, and clinical symptoms such as diarrhea. The survival rate of each group was calculated.

[0060] Finally, after the challenge observation period, all surviving mice were euthanized, and intestinal tissue samples such as small intestine and colon were collected. One part was immediately fixed in 4% paraformaldehyde or 10% neutral formalin solution for subsequent histopathological examination (H&E staining) to assess the protective effect of the vaccine against pathological damage to the intestinal mucosa; another part of the tissue was used for homogenization and plate counting to assess the number of ETEC colonizations in the intestine.

[0061] Results: See Figure 5 In the mouse ETEC infection challenge test, the low-dose group (2×10) 5 Gross anatomical examination of mice (CFU / mouse) revealed normal intestinal tissue morphology and color, with no obvious lesions or congestion; the perianal area was clean, with no fecal residue or diarrhea traces; the medium-dose group (1×10) 6 In mice treated with CFU / mouse, the intestinal wall was thinned and increased in transparency, the intestinal tract was significantly distended, diffuse congestion and reddening of the intestinal segments were observed, and a small amount of loose stool residue was visible around the anus; in the high-dose group (2×10⁻⁶), the intestinal wall was thinned and increased in transparency, the intestinal tract was significantly distended, diffuse congestion and reddening of the intestinal segments were observed 6 Yellow, foamy exudate was visible in the abdominal cavity of mice (CFU / mouse), the intestinal segment was significantly congested and red, and a large amount of watery feces were attached to the perianal area. Figure 5 (A) The diarrhea situation in the early stage of infection of each group of animals was evaluated by fecal characteristics score. Mice in the high-dose group had severe diarrhea, and the degree of diarrhea was relieved as the dose was reduced. Figure 5 (Medium B). Furthermore, observation of mouse body weight and survival rate over 7 consecutive days showed that: the body weight of all three groups of mice showed a significant decreasing trend on day 1; the low-dose group mice rapidly recovered and remained stable on day 2, and all animals survived to the end of the experiment, with a survival rate of 100%; the medium-dose group mice continued to lose body weight until day 4, with only one animal surviving, a survival rate of 25%; all mice in the high-dose group died on day 2, with a survival rate of 0%. Figure 5 (C, D) The median lethal dose (LD50) was calculated using the modified Koch method. 50 This strain has an LD50 (median lethal dose) in mice. 50 ≈1.19×10 6 CFU / each.

[0062] See Figure 6Indirect ELISA was used to detect IgG, IgG1, IgG2a, and IgA antibodies in mice of different groups. The results showed that the IgG antibody levels in the PBS group and the OMVs-WT group were low and not significantly different. After two booster immunizations, the level of SsIE-specific IgG antibodies produced in the OMVs-SsIE vaccine group was significantly higher than that at the time of the first immunization. Meanwhile, the antibody level in the 6P-1-SsIE protein group was slightly increased, but still lower than that in the OMVs-SsIE vaccine group. Figure 6 (B) Antibody detection results for IgG2a and IgG1 were similar to those for IgG; antibody levels in the PBS group and the OMVs-WT group were extremely low and showed no significant difference. After two booster immunizations, the levels of IgG2a and IgG1 antibodies in the OMVs-SsIE vaccine group were significantly increased. Meanwhile, the antibody levels in the 6P-1-SsIE protein group gradually increased, but its ability to induce an immune response was weaker than that in the vaccine group. Figure 6 (C, D). Studies have shown that IgG2a is associated with Th1 immune responses, while IgG1 is associated with Th2 immune responses in mice. Combining the detection results of these two antibodies, it was found that the titer of IgG2a in the OMVs-SsIE immunization group was higher than that of IgG1, proving that OMVs-SsIE vaccine immunization can induce a Th1-biased immune response. The above data show that compared with the PBS group, OMVs-WT group, and 6P-1-SsIE protein group, the OMVs-SsIE vaccine group can induce a stronger humoral immune response. Subsequently, IgA antibodies in each group were detected to evaluate the mucosal immune level in mice. The results showed that the IgA antibody levels in the PBS group and the OMVs-WT group were low and not significantly different; after two booster immunizations, the IgA level in the OMVs-SsIE immunization group increased significantly, while the antibody level in the 6P-1-SsIE protein group only increased slightly after the second booster immunization, indicating that its ability to induce a mucosal immune response was weaker than that of the vaccine group. Figure 6 (E).

[0063] See Figure 7 Eight hours post-infection, the jejunum, ileum, cecum, and colon / rectum were aseptically removed, and ETEC was counted using a plating method. Figure 7 Compared with the PBS group, the bacterial load in the jejunum, ileum, cecum, and colon of mice in the OMVs-SsIE vaccine group was significantly reduced. Although the OMVs-WT group and the 6P-1-SsIE protein group also showed a trend of being lower than the PBS group, the decrease was limited and still higher than the vaccine group. The results indicate that the OMVs-SsIE vaccine group has the highest bacterial clearance capacity, that is, better immune protection for mice. Enteropathogenic Escherichia coli (EPEC) and enteroinvasive Escherichia coli (EIEC) were used to challenge immunized mice. Figure 7(B and C) The results showed that the pathogen load in the intestinal tissues of mice in the OMVs-SsIE vaccine group was still significantly lower than that in other groups, indicating that the vaccine has a good broad-spectrum bacterial clearance ability against different pathogenic Escherichia coli.

[0064] See Figure 8 Eight hours after ETEC infection, paraffin sections (H&E) were collected from the jejunum, ileum, cecum, colon, and rectum of mice in each group for histopathological analysis. The results showed that the intestinal tissue structure in the PBS group was loose, exhibiting severe pathological damage, and no normal tissue morphology was observed. The OMVs-WT group showed a similar overall lesion trend to the PBS group, with specific lesions including: impaired villus integrity in the jejunum and cecum, with single-cell necrosis accompanied by eosinophilic material deposition and some cell debris detachment within the intestinal lumen; a decrease in the number of normal intestinal epithelial cells; mild inflammatory cell infiltration in the ileal mucosa, with some cell debris detachment within the intestinal lumen; and mild single-cell necrosis of intestinal villi accompanied by eosinophilic material deposition in the colon and rectum, with some cell debris detachment within the intestinal lumen. The intestinal tissue morphology in the OMVs-SsIE vaccine group was relatively intact, with clear villus structure, neatly arranged intestinal epithelial cells, and no obvious pathological changes were observed. The 6P-1-SsIE proteome revealed varying degrees of pathological damage, but the overall structure remained relatively intact. Specific lesions included: mild to significant single-cell necrosis of intestinal villi with eosinophilic deposition in the jejunum, ileum, cecum, and colon; mild inflammatory cell infiltration and partial cell debris shedding in the jejunal mucosa; partial cell debris shedding in the ileal lumen; a decrease in the number of normal epithelial cells in the cecum; and mild inflammatory cell infiltration in the colonic mucosa. Figure 8 (A). The tissue damage in each group of mice was then scored. Figure 8 In the PBS and OMVs-WT groups, the scores of the jejunum and ileum in the OMVs-SsIE vaccine group were significantly lower than those in the 6P-1-SsIE protein group, while the scores of the cecum, colon, and rectum in the OMVs-SsIE vaccine group were also lower, but the differences were not statistically significant. These pathological and histological results indicate that OMVs-SsIE provides the best immunoprotective effect against ETEC in the mouse model.

[0065] See Figure 9 To evaluate whether immunized mice could resist lethal doses of ETEC infection, mice were challenged with 2×LD50 virus two weeks after the second booster immunization. 50 The ETEC assay was performed on mice, and their general condition, survival rate, and weight changes were observed and recorded for 7 consecutive days. The results showed that... Figure 9Mice in the PBS group exhibited significant typical clinical symptoms during their survival, including rough coat, lethargy / reduced activity, loss of appetite, abdominal distension, wet and unformed feces, and noticeable anal soiling; all mice ultimately died within 4 days. Mice in the OMVs-WT group showed similar symptoms to the PBS group, and all mice died within 5 days. Mice in the OMVs-SsIE vaccine group initially presented with rough coat, reduced activity, and loss of appetite; their feces were dry and formed; their anal area was clean; their weight gradually recovered starting on day 3; and their survival rate reached 100%. Mice in the 6P-1-SsIE protein group presented with rough coat, reduced activity, and loss of appetite; their feces were wet; and their anal area was slightly soiled; their survival rate was approximately 66%. These results demonstrate that the OMVs-SsIE vaccine can protect mouse models from lethal doses of ETEC.

[0066] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A method for preparing vesicles for delivering SsIE antigen, characterized in that, The method includes: (1) The sequence containing the SsIE antigen is ligated to the linearized plasmid to obtain the recombinant plasmid; (2) Transform the recombinant plasmid into bacteria and screen for positive transformants; (3) The positive transformants were induced to express SsIE antigen and release outer membrane vesicles containing SsIE antigen. (4) Collect and purify the outer membrane vesicles containing SsIE antigen to obtain the product.

2. A nano-vaccine, characterized in that, The nanovaccine comprises vesicles capable of delivering SsIE antigens; preferably, the vesicles delivering SsIE antigens are bacterial extravesicles.

3. A bacterial outer membrane vesicle, characterized in that, The outer surface of the bacterial outer membrane vesicles contains SsIE antigens.

4. The bacterial outer membrane vesicle according to claim 3, characterized in that, The SsIE antigen is localized and displayed on the vesicle surface using a signal peptide and a transmembrane anchoring sequence; preferably, the signal peptide is derived from the Lpp protein and the transmembrane anchoring sequence is derived from the OmpA protein.

5. A bacterial outer membrane vesicle, characterized in that, The bacterial outer membrane vesicles contain a fusion protein Lpp-OmpA-SsIE located on the surface, and the amino acid sequence of the fusion expressed protein Lpp-OmpA-SsIE is shown in SEQ ID NO.

2.

6. A nucleotide sequence encoding the fusion protein Lpp-OmpA-SsIE, characterized in that, The sequence is shown in SEQ ID NO.

1.

7. A fusion protein Lpp-OmpA-SsIE, characterized in that, The amino acid sequence of the fusion protein Lpp-OmpA-SsIE is shown in SEQ ID NO.

2.

8. The nanovaccine according to claim 2, characterized in that, The nanovaccine comprises bacterial exovesicles containing the fusion protein Lpp-OmpA-SsIE on their surface, the amino acid sequence of which is shown in SEQ ID NO.

2.

9. A vector plasmid, characterized in that, The vector plasmid contains the nucleotide sequence of the fusion protein Lpp-OmpA-SsIE as shown in SEQ ID NO.

1.

10. A recombinant bacterium, characterized in that, The recombinant bacteria contain the vector plasmid as described in claim 9.

11. A composition, characterized in that, The composition contains the bacterial outer membrane vesicles of any one of claims 3-5, the nucleotide sequence of claim 6, the fusion protein of claim 7, the vector plasmid of claim 9, or the recombinant bacteria of claim 10.

12. The use of the bacterial outer membrane vesicles according to any one of claims 3-5, the nucleotide sequence according to claim 6, the fusion protein according to claim 7, the vector plasmid according to claim 9, or the recombinant bacteria according to claim 10 in the preparation of a drug, characterized in that, The drug is a drug for the prevention or treatment of Escherichia coli infection and related diseases; preferably, the drug is a vaccine.

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