Preparation method and application of salmonella choleraesuis bionic vesicle for oral delivery of double antigens

Abstract

The invention discloses a preparation method and application of salmonella choleraesuis bionic vesicles capable of being used for oral delivery of double antigens, and relates to the field of vaccines.Lipid A of a salmonella choleraesuis attenuated strain SC014 is modified through a genetic engineering technology, low-endotoxin salmonella choleraesuis engineering bacteria are obtained, then SpyCatcher and SpG are expressed on the surfaces of the salmonella choleraesuis engineering bacteria at the same time through ClyA, and the salmonella choleraesuis bionic vesicles are obtained. According to the present invention, the subunit vaccine model GDH-SpyTag and gD-Fc of the clinically common mixed infection pathogen Streptococcus suis and Pseudorabies virus (PRV) can be respectively coupled, and the negative electricity characteristic of the surface of the subunit vaccine model GDH-SpyTag and gD-Fc are used to wrap the subunit vaccine model GDH-SpyTag and gD-Fc by using the cationic compound COS so as to protect the antigen from being damaged by the gastrointestinal tract environment after the oral immunization; and the presentation of the antigen to digestive tract mucosa immune cells is promoted.

Description

Technical Field

[0001] This invention belongs to the field of vaccines, specifically relating to a method and application for preparing biomimetic vesicles of Salmonella choleraesuis for oral delivery of dual antigens. Background Technology

[0002] Bacterial and viral infectious diseases are significant factors threatening swine health and causing substantial losses in the pig farming industry. Mixed bacterial and viral infections are common in intensive pig farms, and intramuscular vaccination is currently one of the main methods for disease control in pig farms. Subunit vaccines are favored due to their high safety, simple production process, and low cost. However, due to their low immunogenicity, adjuvants are often added and multiple immunizations are required to achieve a control effect. Furthermore, repeated intramuscular injections can easily cause stress in pigs, thus affecting their production performance. Oral immunization with multivalent vaccines can reduce the number of immunizations, improve pig compliance, and induce a mucosal immune response, producing secretory immunoglobulin A (sIgA) on the surface of the mucous membranes to prevent pathogen invasion. However, the acidic environment and enzymes of the digestive system easily destroy antigens, reducing bioavailability; currently, there are no commercially available oral subunit vaccines. Therefore, there is an urgent need to develop a multivalent vaccine delivery system suitable for oral immunization.

[0003] Outer membrane vesicles (OMVs) have shown significant potential for vaccine delivery. OMVs are non-replicating nanoscale vesicles naturally released by Gram-negative bacteria, possessing adjuvant activity and capable of inducing broad-spectrum immune responses. Through genetic engineering, SpyCatcher / SpyTag or SpG / Fc can be used as plug-and-play antigen display modules, allowing specific antigens or nucleic acid-binding molecules to be displayed on the OMV surface, enhancing the immune response and making it an ideal vaccine platform. In 2013, OMVs of meningococcal type B were approved for use in Europe, indicating the broad application prospects of OMVs in vaccine development. However, the low yield of naturally released OMVs from bacteria and the limited capacity of OMVs to load heterologous antigens hinder their further application in vaccine delivery.

[0004] Recent studies have discovered that bacterial biomimetic vesicles (BBVs) with a structure similar to OMV can be prepared from Gram-negative bacteria using high-pressure homogenization technology. The yield of BBVs is 100 times that of OMVs, and their capacity to load heterologous antigens is 30 times higher, effectively addressing the limitations of OMVs as a vaccine delivery platform. Furthermore, due to the negative charge on their surface, BBVs can encapsulate cationic polymers such as chitosan oligosaccharides (COS) on their surface through electrostatic adsorption, enhancing the stability of BBVs and their loaded heterologous antigens in the gastrointestinal tract and improving bioavailability.

[0005] On the other hand, *Salmonella choleraesuis* is a Gram-negative bacterium that primarily infects the host through the intestinal mucosa. Its outer membrane contains adhesion factors that allow it to specifically adhere to the surface of the host's intestinal mucosal epithelial cells. Furthermore, its surface is rich in pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides and flagellin, which can be recognized by pattern recognition receptors on the surface of immune cells, such as dendritic cells in intestinal mucosal epithelial cells and intestinal mucosa-associated lymphoid tissue. This provides conditions for antigen presentation to gastrointestinal mucosal immune cells and activation of the body's adaptive immune response, making it an ideal parent bacterium for oral vaccine development. Therefore, chitosan oligosaccharide-encapsulated *Salmonella choleraesuis* BBV holds promise as a highly efficient oral vaccine delivery platform. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for preparing and applying biomimetic vesicles of *Salmonella choleraesuis* for oral delivery of dual antigens. This invention significantly reduces the endotoxin activity of CSS-BBV by knocking out the tolB, msbB, and pagP genes related to lipid A synthesis and inserting the dephosphatase gene lpxE and the deacylase gene pagL. This solves the problem of high endotoxin content in bacterial biological products. This invention also uses chitosan oligosaccharide (COS) to coat the surface of *Salmonella choleraesuis* biomimetic vesicles (CSS-BBV), forming a positively charged protective layer, which significantly improves the stability of the antigen in simulated gastric and intestinal fluids. This solves the problems of easy degradation and low bioavailability of traditional oral vaccines in the gastrointestinal environment. Furthermore, this invention utilizes the natural intestinal mucosal affinity and immune activation capabilities of *Salmonella choleraesuis*, achieving targeted conjugation of dual antigens (such as GDH and gD) through a surface display system, inducing the body to produce high levels of specific IgG and sIgA antibodies. This solves the problems of weak immunogenicity and difficulty in inducing systemic and mucosal immune responses in oral vaccines.

[0007] The present invention provides a method for preparing biomimetic vesicles of Salmonella choleraesuis, which comprises the following steps:

[0008] Step 1: Clone ClyA, SpG, and 6×His into the pCold I linearized vector to obtain the pCold I-ClyA-SpyCatcher-6×His plasmid; clone ClyA, SpG, and 6×His into the pCold I linearized vector to obtain the pCold I-ClyA-SpG-6×His plasmid; Step 2: Combine the pCold I-ClyA-SpyCatcher-6×His plasmid with the pCold I linearized vector to obtain the pCold I-ClyA-SpG-6×His plasmid. The I-ClyA-SpG-6×His plasmid was co-transformed into SC-L3 competent cells to obtain SC-L3-ClyA-SpyCatcher / SpG recombinant bacteria; the SC-L3 was obtained by modifying the SC014 strain: 1) Using the genomic sequence of the SC014 strain as a template, PCR amplification was performed using DN-1F / DN-1R and DN-2F / DN-2R primers to obtain upstream and downstream fragments lacking the tolB, msbB, and pagP genes, which were named N-up and N-down, respectively; the N-up and N- In the down sequence, N represents the tolB, msbB, and pagP genes; the DN-1F primers are: DtolB-1F: 5'-ttctagtacgtagacacgaatggtcagccatcgaaaga-3'; DmsbB-1F: 5'-ttctagtacgtagacaccgcagcggcacggcctatctg-3'; DpagP-1F: 5'-ttctagtacgtagacactatcccttaatgatgtagcgc-3'

[0009] The DN-1R primers are: DtolB-1R: 5'-ttaattattacatatctcccatacctgggc-3'; DmsbB-1R: 5'-ctggaaaagcctagtcctgatataggttgac-3'; DpagP-1R: 5'-cccttccccgaccgttcaaaaattcgactg-3'

[0010] The DN-2F primers are: DtolB-2F: 5'-gggagatatgtaataattaattgattacta-3'; DmsbB-2F: 5'-tcaggactaggcttttccagggtctgctgac-3'; DpagP-2F: 5'-tttgaacggtcggggaagggcattgttcag-3'

[0011] The DN-2R primers are: DtolB-2R: 5'-ggTAATACGTAGACACTacgcctttaccctgcaggta-3';

[0012] DmsbB-2R: 5'-ggTAATACGTAGACACTatggcgaggtcgtggtcgct-3'; DpagP-2R: 5'-ggTAATACGTAGACACTaacccgccgaaagggcgggtt-3';

[0013] The SC014 strain is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO. 33887 and deposit date of May 8, 2025.

[0014] 2) Using 112-F / 112-R primers and the suicide plasmid pRE112 as a template, PCR amplification was performed to obtain a linear suicide plasmid vector fragment, which was purified and recovered, and named 112-V. Then, N-up and N-down were ligated to 112-V. After correct sequencing, the plasmids were extracted to obtain suicide plasmids P1, P2, and P3, respectively. The 112-F primer was 5'-AGTGTCTACGTATTAccgggaattcatgcag-3'; the 112-R primer was 5'-gtgtctacgtactagaagaagcttg-3'.

[0015] 3) Using primers DpagP-P2-F / R and DmsbB-P3-F / R, linear fragments of P2 and P3 were amplified from the constructed P2 suicide plasmid and P3 suicide plasmid, respectively. Primers DlpxE-F / R and DpagL-F / R were used to amplify the lpxE and pagL fragments from plasmid pQK0055 containing lpxE and pagL fragments. The lpxE fragment was ligated to the P2 linear fragment to obtain the P4 plasmid. The pagL fragment was then ligated to… P3 linear fragment ligation yielded P5 plasmid; the DpagP-P2-F primer was 5'-cggggaagggcattgttcag-3'; the DpagP-P2-R primer was 5'-accgttcaaaaattcgactgtg-3'; the DmsbB-P3-F primer was 5'-gcttttccagggtctgctgacgc-3'; the DmsbB-P3-R primer was 5'- ctagtcctgatataggttgacag-3'; DlpxE-F primer is 5'-GCACGCCGTTAACCCGAGA-3'; DlpxE-R primer is 5'-AGCCACAAATGCTGCTGTGTGG-3'; DpagL-F primer is 5'-agcttccccggataaccagaagcaataaaaaagc-3'; DpagL-R primer is 5'-atcctgttgtgctaaataatctcacgattgc-3';

[0016] 4) P1, P4, and P5 plasmids were transformed into competent cells, and after culture, the bacterial solutions were added to LB plates for culture. After chloramphenicol resistance selection, strains with recombinant P1, P4, and P5 plasmid DNA were obtained. After being plated on LB plates containing 10% sucrose and cultured, single clones were picked and selected for chloramphenicol resistance selection on LB plates. Colony PCR identification using primers DtolB-1F / DtolB-2R showed positive results, which yielded the SCΔtolBΔpagPΔmsbB::lpxE::pagL mutant, named SC-L3.

[0017] Step 3: After expanding the SC-L-CSS recombinant bacteria, IPTG was added for induction, followed by Na2EDTA. The culture was continued, centrifuged, the precipitate was collected, washed, and the bacteria were driven to bud through the gap using an ultra-high pressure homogenizer. The sample passing through the gap was collected, centrifuged, the supernatant was collected, filtered, and CSS-BBV was obtained by molecular sieve chromatography.

[0018] Step 4: Conjugate CSS-BBV with an antigen fused with SpyTag and Fc to obtain CSS-BBV conjugated with dual antigens fused with SpyTag and Fc.

[0019] Step 5: Add the chitosan oligosaccharide solution dropwise to the CSS-BBV coupled with SpyTag and Fc dual antigens. After standing, remove the excess chitosan oligosaccharide to obtain the chitosan oligosaccharide-coated CSS-BBV loaded with dual antigens.

[0020] Furthermore, the method for preparing SC-L3 competent cells is as follows:

[0021] 1) Resuscitate SC-L3 from a -80℃ freezer and incubate overnight in a medium containing Amp antibiotic. The next day, transfer the bacterial culture to LB liquid medium and incubate until the OD600 of the bacterial culture is 0.3~0.7. 2) Centrifuge the bacterial culture at 4℃, discard the supernatant and collect the precipitate. 3) Add pre-cooled 0.1 M CaCl2 solution to the collected precipitate, incubate on ice, centrifuge and collect the precipitate. 4) Repeat step 3) once. 5) Add pre-cooled 0.1 M CaCl2 solution containing 10% glycerol, mix well and store at -80℃.

[0022] Furthermore, the IPTG induction involves adding IPTG to a final concentration of 0.1 mM and inducing culture at 13-17 ℃ for 5-8 hours.

[0023] Furthermore, the addition of Na2EDTA involves adding 1-3 mM Na2EDTA and continuing incubation for 2 hours.

[0024] Furthermore, the washing described in step three is performed using PBS buffer.

[0025] Furthermore, the conditions for the ultra-high pressure homogenizer to drive bacteria to bud through the gaps in step three are 1000~1500 bar, 2~4 times, 4℃.

[0026] Furthermore, the conjugation of CSS-BBV with the antigen fused with SpyTag and Fc in step four is specifically as follows:

[0027] First, the CSS-BBV and the antigen of the fusion SpyTag were mixed and stirred in a buffer solution at 4°C for 1–1.5 hours. Then, the uncoupled fusion SpyTag antigen was removed using a 100 kDa ultrafiltration centrifuge tube to obtain the CSS-BBV with the fusion SpyTag conjugated. Second, the CSS-BBV with the fusion SpyTag conjugated and the antigen of the fusion Fc were mixed and stirred in a phase buffer solution at 4°C for 1–1.5 hours. Then, the uncoupled fusion Fc antigen was removed using a 100 kDa ultrafiltration centrifuge tube to obtain the CSS-BBV with the fusion SpyTag and Fc dual antigen conjugated. The buffer solution was prepared by mixing 20 mM Tris-HCl and 50 mM NaCl, with a pH of 8.0.

[0028] Furthermore, the mass concentration of chitosan oligosaccharide in step five is 2~14 mg / mL, and the pH value is 5.0~6.0.

[0029] Furthermore, the standing time described in step five is 30-60 minutes at 4°C.

[0030] The biomimetic vesicles of Salmonella choleraesuis prepared according to the present invention are used to prepare oral delivery dual antigens.

[0031] The present invention has the following beneficial effects:

[0032] This invention significantly reduces the endotoxin content of CSS-BBV through low-endotoxin modification, thereby reducing the secretion of inflammatory factors (IL-6, TNF-α) in mice after immunization, lowering the immune stress response, and improving vaccine safety. Utilizing two antigen-conjugation systems, SpyCatcher / SpyTag and SpG / Fc, two different antigens are directionally conjugated on the surface of CSS-BBV, ensuring efficient co-delivery of the two antigens and maintenance of immunogenicity. Encapsulating CSS-BBV with chitosan oligosaccharides forms a protective layer, significantly reducing the degradation of antigens by gastric acid and digestive enzymes, and improving antigen survival and bioavailability in the gastrointestinal tract. Oral immunization with chitosan oligosaccharide-coated CSS-BBV carrying two antigens induces high levels of specific IgG and sIgA antibodies in mice. While IgG antibody levels were not significantly different from the intramuscular injection group, sIgA antibody levels were significantly higher than in other oral groups, indicating that this invention significantly enhances the mucosal immune response. Animal experiments showed that orally administered chitosan oligosaccharide-coated dual-antigen CSS-BBV provided 100% protection against lethal doses of Streptococcus suis and pseudorabies virus, significantly superior to existing commercial vaccines and unconjugated antigen mixtures, demonstrating excellent immunoprotective effects. In summary, the biomimetic vesicle vaccine platform for Salmonella choleraesuis constructed in this invention not only solves key problems existing in the prior art but also achieves breakthroughs in the stability, immunogenicity, and safety of oral vaccines, possessing broad application value and promotional significance. Attached Figure Description

[0033] Figure 1 Gene mutation strategy and PCR identification diagram of strain SC014; Figure 2 A schematic diagram illustrating the construction process of GDH-gD-Fc-CSS-BBV vesicles; Figure 3 Expression of ClyA-SpyCatcher and ClyA-SpG in SC-L-CSS(A) and purified CSS-BBV; Figure 4 Identification of GDH-SpyTag and gD-Fc; Figure 5Transmission electron microscope images of CSS-BBV before and after coupling with GDH-SpyTag and gD-Fc; Figure 6 Changes in WB bands before and after CSS-BBV coupling with GDH-SpyTag and gD-Fc; Figure 7 Changes in particle size distribution before and after CSS-BBV coupling with GDH-SpyTag and gD-Fc; Figure 8 Coupling efficiency analysis of CSS-BBV for GDH-SpyTag and gD-Fc; Figure 9 Endotoxin activity analysis of CSS-BBV (A) Quantitative analysis of endotoxins; (B) Levels of IL-6 and TNF-α produced by RAW264.7 cells stimulated by CSS-BBV and SC-BBV; Figure 10 Changes in surface potential of GDH-gD-Fc-CSS-BBV@COS encapsulated with different concentrations of COS; Figure 11 Transmission electron microscope image of GDH-gD-Fc-CSS-BBV@COS; Figure 12 The protective efficacy of COS against GDH-gD-Fc-CSS-BBV was evaluated in vitro using a simulated gastrointestinal fluid environment. Figure 13 Cytotoxicity of GDH-gD-Fc-CSS-BBV@COS encapsulated with different concentrations on RAW264.7 cells; Figure 14 Uptake of biomimetic vesicles by RAW264.7 cells; Figure 15 Changes in body temperature in mice within 12 hours after immunization; Figure 16 Levels of inflammatory factors in mouse serum within 12 hours post-immunization; Figure 17 Specific IgG levels in immunized mice; Figure 18 Specific sIgA levels in immunized mice; Figure 19 PRV neutralizing antibody levels in immunized mice; Figure 20 Clinical scores of mice after challenge with PRV and Streptococcus suis; Figure 21 Survival rate of mice after challenge with PRV and Streptococcus suis. Detailed Implementation

[0034] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0035] Example 1

[0036] 1. Strategies for Low Endotoxin Modification of SC Strains

[0037] To reduce the endotoxin activity of biomimetic vesicles, this invention modifies the parental bacterial strain to reduce endotoxin levels. A suicide plasmid-mediated homologous recombination method is used to knock out and insert genes related to lipid A synthesis, thereby altering the lipid A structure and reducing bacterial endotoxin activity. Using SC014 as the target strain, the tolB, pagP, and msbB genes are deleted, and the dephosphatase gene lpxE and the deacylase gene pagL are inserted at the pagP and msbB deletion sites, respectively. The SC-L3 strain was obtained by modifying the SC014 strain: 1) Using the genomic sequence of the SC014 strain as a template, PCR amplification was performed using DN-1F / DN-1R and DN-2F / DN-2R primers (the amplification system and conditions are shown in Tables 1 and 2), obtaining upstream and downstream fragments of the tolB, msbB, and pagP genes, which were named N-up and N-down, respectively (where N represents the names of the deleted tolB, msbB, and pagP genes); the DN-1F primers were: DtolB-1F: 5'-ttctagtacgtagacacgaatggtcagccatcgaaaga-3'; DmsbB-1F: 5'-ttctagtacgtagacaccgcagcggcacggcctatctg-3'; DpagP-1F: 5'-ttctagtacgtagacactatcccttaatgatgtagcgc-3'

[0038] The DN-1R primers are: DtolB-1R: 5'-ttaattattacatatctcccatacctgggc-3'; DmsbB-1R: 5'-ctggaaaagcctagtcctgatataggttgac-3'; DpagP-1R: 5'-cccttccccgaccgttcaaaaattcgactg-3'

[0039] The DN-2F primers are: DtolB-2F: 5'-gggagatatgtaataattaattgattacta-3'; DmsbB-2F: 5'-tcaggactaggcttttccagggtctgctgac-3'; DpagP-2F: 5'-tttgaacggtcggggaagggcattgttcag-3'

[0040] The DN-2R primers are: DtolB-2R: 5'-ggTAATACGTAGACACTacgcctttaccctgcaggta-3';

[0041] DmsbB-2R: 5'-ggTAATACGTAGACACTatggcgaggtcgtggtcgct-3'; DpagP-2R: 5'-ggTAATACGTAGACACTaacccgccgaaagggcgggtt-3';

[0042] The SC014 strain is Salmonella choleraesuis, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 33887 and deposit date of April 28, 2025.

[0043] 2) Using 112-F / 112-R primers and suicide plasmid pRE112 as a template, PCR amplification was performed to obtain a linear suicide plasmid vector fragment. The PCR product was recovered according to the instructions of the DNA purification and recovery kit (this applies to all DNA recovery processes involved in this invention) and named 112-V. The N-up, N-down and 112-V recovered fragments were ligated at 50°C for 30 min using a seamless cloning kit. The ligation products were transformed into DH5α competent cells, and the bacterial culture was sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Sequencing results were analyzed using SnapGene 3.1.1. Correctly sequenced bacterial cultures were transferred to culture medium at a 1:100 ratio and cultured overnight. Plasmids were extracted using a plasmid extraction kit, yielding suicide plasmids P1 (ΔtolB), P2 (ΔpagP), and P3 (ΔmsbB). The 112-F primer was 5'-AGTGTCTACGTATTAccgggaattcatgcag-3'; the 112-R primer was 5'-gtgtctacgtactagaagaagcttg-3'.

[0044] 3) Using primers DpagP-P2-F / R and DmsbB-P3-F / R, linear fragments of P2 and P3 were amplified from the constructed P2 suicide plasmid and P3 suicide plasmid, respectively (PCR reaction system and reaction conditions are detailed in Tables 1 and 2). Primers DlpxE-F / R and DpagL-F / R were used to amplify the lpxE and pagL fragments from plasmid pQK0055 containing lpxE and pagL fragments (PCR reaction system and reaction conditions are detailed in Tables 1 and 2). (1 and 2) The lpxE fragment was ligated to the P2 linear fragment using a seamless cloning kit to obtain the P4 plasmid, and the pagL fragment was ligated to the P3 linear fragment to obtain the P5 plasmid; the DpagP-P2-F primer was 5'-cggggaagggcattgttcag-3'; the DpagP-P2-R primer was 5'-accgttcaaaaattcgactgtg-3'; the DmsbB-P3-F primer was 5'- gcttttccagggtctgctgacgc-3'; DmsbB-P3-R primer is 5'-ctagtcctgatataggttgacag-3'; DlpxE-F primer is 5'-GCACGCCGTTAACCCGAGA-3'; DlpxE-R primer is 5'-AGCCACAAATGCTGCTGTGTGG-3'; DpagL-F primer is 5'-agcttccccggataaccagaagcaataaaaaagc-3'; DpagL-R primer is 5'-atcctgttgtgctaaataatctcacgattgc-3';

[0045] 4) Taking the mutation of the tolB gene in strain SC014 as an example, the construction methods for other mutations or insertions are similar.

[0046] (1) Preparation of homologous recombinant donor bacteria: The recombinant suicide plasmid P1 constructed above was transformed into competent S17-1λpir cells. The resulting recombinant S17-1λpir strain containing the suicide plasmid is the homologous recombinant donor bacteria, which is stored at -80℃ for later use.

[0047] (2) Conjugation transfer: Resuscitate the SC strain (recipient strain) and the above-mentioned homologous recombinant donor strain from the refrigerator and culture them until the bacterial culture reaches OD. 600The value was approximately 0.8. 600 μL of donor bacteria and 400 μL of recipient bacteria were mixed and added to LB plates and cultured at 37°C tilted upright for 24 h. (3) Chloramphenicol positive selection: The bacterial growth on the plate was re-streaked onto LB plates containing chloramphenicol and cultured overnight at 37°C. Subsequently, the single clones on the plate were streaked again onto LB plates with the same resistance and cultured overnight at 37°C to obtain strains in which the SC genome and recombinant suicide plasmid DNA were integrated. (4) Sucrose negative selection: 3-6 single clones selected from the above chloramphenicol resistance on LB plates were cultured to OD. 600 The concentration was approximately 0.5. The bacterial culture was diluted to 100 and 1000 times with LB medium, and 100 μL was spread on LB plates containing 10% sucrose and incubated at room temperature for 24 h. (5) Identification: Single clones were randomly selected and streaked on LB plates containing and without chloramphenicol. The clones were incubated at 37℃ for 15 h. Clones that did not grow on LB plates containing chloramphenicol and grew well on plates without chloramphenicol were suspected positive clones. Colony PCR was performed using primers DtolB-1F / DtolB-2R. SC strain was used as a negative control. The PCR identification procedure and system reference enzyme usage (2×Taq PCR Mix (KT201)) were used. The size of the PCR product was observed by 1% agarose gel electrophoresis.

[0048] Following the same method, using the constructed suicide plasmids P2, P3, P4 (::lpxE) and P5 (:: pagL), the deletion and insertion of the pagP, msbB, lpxE and pagL genes were introduced on the basis of the tolB gene deletion, resulting in the SCΔtolBΔpagPΔmsbB::lpxE::pagL mutant, named SC-L3. The upstream nucleotide sequence of the tolB gene is shown in SEQ ID No. 1, the downstream nucleotide sequence of the tolB gene is shown in SEQ ID No. 2, the upstream nucleotide sequence of the msbB gene is shown in SEQ ID No. 3, the downstream nucleotide sequence of the msbB gene is shown in SEQ ID No. 4, the upstream nucleotide sequence of the pagP gene is shown in SEQ ID No. 5, the downstream nucleotide sequence of the pagP gene is shown in SEQ ID No. 6, the nucleotide sequence of the tolB gene is shown in SEQ ID No. 7, the nucleotide sequence of the msbB gene is shown in SEQ ID No. 8, the nucleotide sequence of the pagP gene is shown in SEQ ID No. 9, the nucleotide sequence of the lpxE gene is shown in SEQ ID No. 10, and the nucleotide sequence of the pagL gene is shown in SEQ ID No. 11.

[0049] Table 1 PCR amplification system

[0050]

[0051] Table 2 PCR reaction procedure

[0052]

[0053] 2. Construction of recombinant plasmids for displaying heterologous proteins on bacterial surfaces

[0054] To display heterologous proteins on bacterial surfaces, ClyA was expressed in tandem with the target protein. First, the genes encoding ClyA, SpyCatcher / SpG, and 6×His were cloned into the pCold I linearized vector, and three G4S flexible linkers were added after ClyA to construct the pCold I-ClyA-SpyCatcher-6×His and pCold I-ClyA-SpG-6×His plasmids. The coding sequences of the relevant genes were synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd., and the specific amino acid and nucleotide sequences are as follows. The amino acid sequence of ClyA (N-terminal to C-terminal) is shown in SEQ ID No. 16, the nucleotide sequence of ClyA is shown in SEQ ID No. 17, the amino acid sequence of SpyCatcher (N-terminal to C-terminal) is shown in SEQ ID No. 18, the nucleotide sequence of SpyCatcher is shown in SEQ ID No. 19, the amino acid sequence of SpG protein (N-terminal to C-terminal) is shown in SEQ ID No. 20, and the nucleotide sequence of SpG protein is shown in SEQ ID No. 21.

[0055] 3. Construction and induction of recombinant expression strains

[0056] The pCold I-ClyA-SpyCatcher-6×His and pCold I-ClyA-SpG-6×His plasmids successfully constructed above were co-transformed into SC-L3 competent cells to obtain SC-L3-ClyA-SpyCatcher / SpG (SC-L-CSS) recombinant bacteria. The competent cell preparation steps are as follows: (1) SC-L3 cells were revived from a -80℃ freezer and cultured overnight in a medium containing Amp antibiotic. The next day, the bacterial culture was transferred to 100 mL of LB liquid medium at a ratio of 1:100 and cultured under the same conditions until the OD600 of the bacterial culture was about 0.5. (2) The bacterial culture was dispensed into two pre-cooled sterile centrifuge tubes and centrifuged at 3500×g for 10 min at 4°C. The supernatant was discarded and the precipitate was collected. (3) 10 mL of pre-cooled 0.1 M CaCl2 solution was added to each of the two centrifuge tubes and incubated on ice for 20 min. After centrifugation under the same conditions, the precipitate was collected. (4) The above steps were repeated once.

[0057] (5) Add 2 mL of pre-cooled 0.1 M CaCl2 solution containing 10% glycerol to each tube, gently pipette with a sterile pipette tip, and aliquot into 100 μL tubes and store at -80℃. Resuscitate the SC-L-CSS strain and culture overnight. The next day, transfer the strain at a 1:100 ratio, and culture the bacterial solution until the OD600 is about 0.8. Add IPTG to a final concentration of 0.1 mM, induce culture at 15℃ for 6 h, and collect the bacterial solution. Note that the SC-L-CSS strain needs to be cultured in a medium containing both Amp and Kan antibiotics. After centrifugation (12,000×g, 2 min), resuspend in PBS, mix with the loading buffer, boil in water for 10 min, centrifuge, and take 10 μL for loading. Perform SDS-PAGE to confirm the expression of ClyA-SpyCatcher and ClyA-SpG.

[0058] 4. Preparation and purification of CSS-BBV

[0059] Add 2 mM Na2EDTA to the IPTG-induced SC-L-CSS bacterial culture from the previous step and continue culturing for 2 h. Then, collect the precipitate by centrifugation at 10,000 × g for 30 min at 4°C. After washing twice with PBS buffer, use an ultra-high pressure homogenizer (SPCH-10) to drive the bacteria through the gap for budding (conditions: 1200 bar, 3 times, 4°C). Centrifuge the sample through the gap at 9000 × g for 30 min at 4°C, collect the supernatant and filter it through a 0.45 μm filter membrane. Next, separate the filtrate by molecular sieve chromatography to obtain CSS-BBV.

[0060] 5. Expression and purification of GDH-SpyTag and gD-Fc recombinant proteins

[0061] For ease of evaluation, the sequences of the GDH-SpyTag and gD-Fc recombinant proteins were cloned into the pET8a and pCDNA3.1 plasmids, respectively. To facilitate purification, a 6×His fusion was added to the C-short of the insert fragment, thereby constructing pET8a-GDH-SpyTag-His and pCDNA3.1-gD-Fc-His, respectively. pET8a-GDH-SpyTag-His was transformed into *E. coli* DH-5α, single clones were selected, sequenced, and then inoculated into LB medium containing chloramphenicol and cultured at 37°C until OD (digestive activity) was achieved. 600 When the concentration reached approximately 0.6, IPTG was added to a final concentration of 0.1 mM, and the bacterial culture was induced at 16℃ for 20 h. After centrifugation, the bacterial cells were collected, and the supernatant was collected after sonication and centrifugation. The recombinant protein GDH-SpyTag was purified using nickel column affinity chromatography. pCDNA3.1-gD-Fc was transfected into 293T cells. After 48 h, the cell culture medium was collected, centrifuged at 8000 rpm for 20 min at 4℃, and the supernatant was filtered through a 0.22 μm filter. The supernatant was then incubated with nickel column packing material at 4℃ for 2 h, and the recombinant protein gD-Fc was purified using nickel column affinity chromatography. The purification efficiency of recombinant proteins GDH-SpyTag and gD-Fc was identified by SDS-PAGE and Western blotting, respectively. The amino acid sequence of GDH-SpyTag-His is shown in SEQ ID No. 12 of the sequence listing. The nucleotide sequence of GDH-SpyTag-His is shown in SEQ ID No. 13 of the sequence listing. The gD-Fc amino acid sequence is shown in the sequence listing SEQ ID No. 14.

[0062] The gD-Fc nucleotide sequence is shown in the sequence listing SEQ ID No. 15.

[0063] 6. Coupling of CSS-BBV with GDH-SpyTag and gD-Fc

[0064] The CSS-BBV conjugation of dual antigens is performed in two steps. First, the CSS-BBV and GDH-SpyTag obtained above are mixed in a specific ratio in a buffer solution (20 mM Tris-HCl, 50 mM NaCl, pH 8.0) at 4°C for 1 hour. Then, the unconjugated GDH-SpyTag is removed using an ultrafiltration centrifuge tube with a 100 kDa molecular weight cutoff, yielding GDH-CSS-BBV. The unconjugated GDH-SpyTag is detected using the BCA method, and the conjugation efficiency between GDH-SpyTag and CSS-BBV is calculated using the following formula: Conjugation efficiency (%) = (Feed amount - Residual amount) / Feed amount × 100%.

[0065] Next, GDH-CSS-BBV and gD-Fc are mixed under the same conditions and unbound gD-Fc is removed to obtain GDH-gD-Fc-CSS-BBV. The coupling efficiency of gD-Fc and GDH-CSS-BBV is calculated using the same method as in the previous step.

[0066] 7. Preparation of GDH-gD-Fc-CSS-BBV@COS

[0067] Chitosan oligosaccharide (COS) was dissolved in deionized water to prepare COS solutions of different concentrations (2, 4, 6, 8, 10, 12, and 14 mg / mL), and the pH was adjusted to 5.0–6.0. ​​While stirring, the COS solutions of different concentrations were slowly added dropwise to the above GDH-gD-Fc-CSS-BBV solution. The mixture was allowed to stand at 4°C for 30 min, and then excess COS was removed using an ultrafiltration centrifuge tube with a 100 kDa molecular weight cutoff, yielding GDH-gD-Fc-CSS-BBV@COS.

[0068] 8. Characterization of BBV

[0069] The morphology, particle size, and surface potential of CSS-BBV, GDH-gD-Fc-CSS-BBV, and GDH-gD-Fc-CSS-BBV@COS were detected using TEM and a NanoZS90 nanoparticle size analyzer. Simultaneously, GDH-CSS-BBV and GDH-gD-Fc-CSS-BBV were identified using immunoelectron microscopy. The methods are as follows:

[0070] Immunoelectron microscopy: After binding to each antigen, unadsorbed proteins were removed using a centrifugal ultrafiltration device with a molecular weight cutoff of 100 kDa. The cutoff samples GDH-CSS-BBV and GDH-gD-Fc-CSS-BBV were incubated with rabbit-derived GDH and mouse-derived gD polyclonal antibodies, respectively, and then labeled with corresponding 10 nm and 5 nm gold-labeled antibodies. TEM was used to observe the antigen binding on the vesicle surface. Particle size and surface potential determination: The samples were diluted to an appropriate concentration (usually 0.1–1 mg / mL) and filtered through a 0.45 µm filter membrane before being loaded into cuvettes, avoiding bubble formation. The cuvettes were placed in a preheated NanoZS90 sample cell, and particle size and surface potential were determined according to the instrument's operating instructions. The endotoxin content of CSS-BBV was detected using a LAL colorimetric reagent kit. 10 µL of endotoxin detection reagent solution and the sample to be tested were added to a microcentrifuge tube. Subsequently, 10 µL of colorimetric reagent solution was added, and the tubes were incubated at 37ºC in the dark for 9 min. Next, add 50 µL of Buffer A, Buffer B, and Buffer C sequentially, let stand for 5 min, and then measure the absorbance at 545 nm using a microplate reader. Mix thoroughly after adding each reagent.

[0071] 9. In vitro simulated gastrointestinal digestion experiment

[0072] First, commercially available simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were preheated at 37°C. Then, GDH-gD-Fc-CSS-BBV or GDH-gD-Fc-CSS-BBV@COS samples were added to SGF at a 1:10 ratio, or to SIF at a 1:1 ratio. Next, the mixture was incubated at 37°C, and SDS-PAGE analysis was performed at different time points within 1 hour. Protein degradation was calculated based on changes in grayscale values.

[0073] 10. Cytotoxicity assay of GDH-gD-Fc-CSS-BBV@COS

[0074] The cytotoxicity of GDH-gD-Fc-CSS-BBV@COS was detected using the CCK-8 assay. RAW264.7 cells were digested and resuspended in complete culture medium at a concentration of 1×10⁻⁶. 4Cells were seeded at a density of 100 μL per well in a 96-well plate and cultured at 37°C in a 5% CO2 incubator until confluence reached 90%. A blank control (culture medium only, no cells) and a control group (cells, no treatment) were also included. The culture medium was removed from the plates, and 100 µL of GDH-gD-Fc-CSS-BBV@COS coated with different concentrations of COS was added to each well, with three replicates for each concentration. After 24 h of culture, the culture medium was replaced, and 10 µL of CCK-8 reagent was added to each well (avoiding air bubbles). The cells were cultured for another 2 h, and the absorbance (OD) of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using the formula: Cell viability (%) = (Experimental group OD - Blank group OD) / (Control group OD - Blank group OD) × 100%.

[0075] 11. Study on the internalization capacity of GDH-gD-Fc-CSS-BBV@COS by macrophages

[0076] CSS-BBV, GDH-CSS-BBV@COS, and GDH-gD-Fc-CSS-BBV@COS were labeled with a lipophilic fluorescent dye (DiD) according to the instructions. Subsequently, the cells were washed twice with PBS and ultracentrifuged at 150,000×g (4℃) for 2 h to remove unbound dye, obtaining DiD-labeled vesicles. These vesicles were then coated with COS to study the internalization of CSS-BBV, GDH-CSS-BBV@COS, and GDH-gD-Fc-CSS-BBV@COS in mouse monocyte-macrophage RAW264.7 cells. 5 The cells were seeded at a density of approximately 70% in 6-well plates and cultured until confluence reached about 70%. Then, 3 μg of CSS-BBV, GDH-CSS-BBV@COS, and GDH-gD-Fc-CSS-BBV@COS were added to the cells, and the cells were incubated for 16 h. Subsequently, the cells were incubated at 37°C for 10 min using CellMask™ green plasma membrane staining agent and NucBlue™ live cell staining agent ReadyProbes™. After washing twice with PBS and replacing with fresh culture medium, the cells were observed using a laser confocal microscope (LSM980-ZEISS).

[0077] 12. Evaluation of the immunogenicity of GDH-gD-Fc-CSS-BBV@COS

[0078] One hundred and ten 6-week-old SPF-grade female BALB / c mice were randomly divided into 11 groups: PBS injection group, gD-Fc injection group, GDH-SpyTag injection group, inactivated streptococcal vaccine injection group, PRV inactivated vaccine injection group, CSS-BBV+GDH+gD-Fc+COS injection group, GDH-gD-Fc-CSS-BBV@COS injection group, gD-Fc oral administration group, GDH-SpyTag oral administration group, CSS-BBV+GDH+gD-Fc+COS oral administration group, and GDH-gD-Fc-CSS-BBV@COS oral administration group. The CSS-BBV+GDH+gD-Fc+COS group consisted of a mixture of CSS-BBV, GDH, gD-Fc, and COS without conjugation. The gD-Fc injection and GDH-SpyTag injection groups involved emulsifying gD-Fc or GDH-SpyTag with ISA201 adjuvant at a 1:1 volume ratio. The gD-Fc oral and GDH oral groups did not require adjuvant addition. The antigen immunization standard was 30 μg per mouse. The inactivated streptococcal vaccine (Collining, Wuhan Keqian) and PRV inactivated vaccine (Keweining, Wuhan Keqian) were administered at 100 μL per mouse in each group (Table 3). Each group of mice underwent two immunizations, two weeks apart, with the initial immunization date recorded as day 0. To assess the endotoxin activity of each group's immunized samples, body temperature was measured at different time points (1, 3, 6, and 12 h) within 12 h after the initial immunization. Simultaneously, serum samples were collected from mice at the same time point, and the levels of IL-6 and TNF-α in the serum were detected using a commercially available ELISA kit, following the instructions. Body weight changes in each immunized group were continuously monitored for 2 weeks post-immunization. Blood and vaginal secretions were collected from each immunized mouse before the initial immunization and at weeks 1, 2, 3, 4, and 5 post-immunization. Indirect ELISA was used to detect the levels of anti-LPS, OMPs, GDH, and gD IgG in mouse serum and anti-sIgA antibodies in vaginal secretions. For mice immunized with gD, gD neutralizing antibodies in their serum were detected, following the steps below: 100 μL Vero E6 cells (10... 5 Mouse serum (cells / mL) was seeded into 96-well plates and cultured at 37°C and 5% CO2 until 80-90% complete cell proliferation. The serum was inactivated at 56°C for 30 min and then serially diluted 2-fold using DMEM medium. 50 μL of the diluted serum was then mixed with an equal volume of 200 TCID50. 50PRV virus solution was mixed and incubated at 37°C and 5% CO2 for 1 h. Then, 100 μL of the virus-serum mixture was seeded into Vero E6 cells in 96-well plates to observe specific cytopathic effects (CPE). Pure virus, diluted serum, and blank cell control groups were also included. All serum dilutions were performed in quadruplicate. The titer of the highest serum dilution resulting in 50% PRV infection inhibition was recorded as the serum neutralizing titer. Neutralizing antibodies were calculated using the Reed-Muench method. Five weeks after initial immunization, vaccinated and unvaccinated mice (n=6 / group) were challenged with lethal doses (a challenge model had been established in the laboratory) of *S. suis* strain 2 and PRV strain. The health status of the mice was then assessed and survival rates were calculated within 2 weeks post-challenge.

[0079] Experimental results

[0080] 1. Identification and characterization of SC mutant strains

[0081] Using SC014 as the background strain, suicide plasmids P1, P2, P3, P4, and P5 were used to perform deletions of the tolB, msbB, and pagP genes and insertions of the lpxE and pagL genes. Figure 1 A). To confirm the successful construction of the mutant strain, primers DtolB-1F / 2R, DpagP-1F / 2R, and DmsbB-1F / 2R were used to amplify relevant gene fragments using the SC genome as a template. Theoretically, the PCR product sizes using SC014 (abbreviated as SC) as a template are approximately 2220, 1555, and 1911 bp, respectively. If the strain successfully mutates, the PCR product using the corresponding strain's genome as a template will reduce the size of each gene. The known gene sizes for tolB, pagP, and msbB are 1293, 573, and 969 bp, respectively. Figure 2 As shown in B, the PCR products were between 800-1000 bp in size. Furthermore, gene insertion was identified using primers specific to lpxE and pagL, and the PCR products were as expected (see Figure B). Figure 1 (C, D). These results all indicate that the deletion and insertion of the target gene were successful.

[0082] 2. CCS-BBV can be coupled to GDH-SpyTag and gD-Fc simultaneously.

[0083] To verify the successful construction of the SC-L3-ClyA-SpyCatcher / SpG (SC-L-CSS) recombinant bacteria, SDS-PAGE was used to analyze the expression effects of ClyA-SpyCatcher and ClyA-SpG in SC-L-CSS. The results are as follows: Figure 2As shown in Figure A, the molecular weights of ClyA-SpyCatcher and ClyA-SpG successfully expressed by the recombinant strain were approximately 48 kDa and 57 kDa, respectively, consistent with theoretical values. Subsequently, SDS-PAGE analysis was performed on CSS-BBV before and after purification. The results are as follows... Figure 2 As shown in Figure B, molecular sieve chromatography can effectively separate CSS-BBV from other proteins. To facilitate evaluation of the conjugation of CSS-BBV to two pattern antigens (GDH-SpyTag and gD-Fc), GDH-SpyTag and gD-Fc were identified by SDS-PAGE and Western blotting analysis. The results are as follows: Figure 3 As shown, the molecular weights of GDH-SpyTag and gD-Fc are approximately 50 kDa and 100 kDa, respectively. To achieve the targeted conjugation of CSS-BBV with the two antigens, a stepwise strategy was adopted: first, CSS-BBV was reacted with GDH-SpyTag, and then gD-Fc was further introduced, ultimately obtaining the GDH-gD-Fc-CSS-BBV complex vesicles. Figure 4 TEM showed that CSS-BBV appeared as uniform, round vesicles; immunogold labeling results clearly revealed the stepwise coupling process: 10 nm gold particles were visible on the surface of GDH-CSS-BBV (blue arrows), while 10 nm and 5 nm gold particles were simultaneously detected on the surface of GDH-gD-Fc-CSS-BBV (red arrows), visually confirming the successful loading of the two antigens. Figure 5 Western blot analysis also showed that as the coupling process progressed, the vesicle bands gradually shifted upwards, and the molecular weight increased ( ). Figure 6 Particle size analysis further confirmed that the particle sizes of CSS-BBV (94.91 nm ± 0.96 nm, PDI = 0.294 ± 0.003), GDH-CSS-BBV (144.87 nm ± 1.69 nm, PDI = 0.214 ± 0.021) to GDH-gD-Fc-CSS-BBV (195.57 nm ± 1.14 nm, PDI = 0.214 ± 0.18) increased in a gradient, and the distribution was uniform. Figure 7 Quantitative results show that the maximum coupling efficiency between CSS-BBV and GDH-SpyTag is 55.83% ± 0.05%, and the maximum coupling efficiency between GDH-CSS-BBV and gD-Fc is 69.92% ± 0.02%. These results demonstrate that GDH and gD-Fc can be simultaneously coupled to CSS-BBV. Figure 8 To ensure the biosafety of BBV, the endotoxin content of CSS-BBV and the inflammatory factors IL-6 and TNF-α induced by RAW264.7 cells were measured. The results are as follows: Figure 9 As shown in Figure A, CSS-BBV has a lighter red color than SC-BBV prepared from wild-type SC. Quantitative analysis showed that the endotoxin content of CSS-BBV was approximately 0.48 EU / mL, significantly lower than that of SC-BBV. Subsequently, RAW264.1 cells were incubated with CSS-BBV and SC-BBV, respectively, to assess changes in the levels of pro-inflammatory cytokines produced by cyst-stimulated cells. The results are as follows. Figure 9 As shown in Figure B, both CSS-BBV and SC-BBV significantly induced the production of IL-6 and TNF-α, but the production level of CSS-BBV was significantly lower than that of SC-BBV. These results indicate that CSS-BBV prepared from lipid A-modified SC-L3 has lower endotoxin activity, which meets application requirements.

[0084] 3. GDH-gD-Fc-CSS-BBV can be modified with a COS coating.

[0085] To evaluate the effect of COS concentration on the modification effect of GDH-gD-Fc-CSS-BBV coating, the surface potential of GDH-gD-Fc-CSS-BBV@COS prepared with different COS concentrations was measured. The results showed that as the COS concentration increased from 0 mg / mL to 14 mg / mL, the vesicle surface potential increased from -44.4 mV ± 2.5 mV to +10.18 mV, and there was a positive correlation with the COS concentration. Figure 10 This initially indicates that CCS-OMV bound to the dual antigens can still be effectively encapsulated by COS. Furthermore, TEM observation of the surface morphology of GDH-gD-Fc-CSS-BBV@COS after COS coating revealed black chitosan oligosaccharide particles deposited on the vesicle surface (red arrows). Figure 11 This provides more intuitive evidence that COS is wrapped around the surface of GDH-gD-Fc-CSS-BBV.

[0086] 4. Evaluation of the protective effect of COS against GDH-gD-Fc-CSS-BBV in an in vitro simulated gastrointestinal fluid environment.

[0087] To verify the protective effect of the COS coating on GDH-gD-Fc-CSS-BBV, the degradation of GDH-gD-Fc-CSS-BBV with and without COS coating protection at 37°C was analyzed in vitro under simulated gastrointestinal fluid (SGF and SIF) conditions. The results are as follows: Figure 12As shown, without COS protection, GDH-gD-Fc-CSS-BBV degrades rapidly, with its integrity in SGF and SIF dropping sharply to 12.1% and 8.3%, respectively, within 2 minutes. After COS coating modification, the antigen integrity of GDH-gD-Fc-CSS-BBV@COS was maintained at 80.3% and 60.3% in SGF and SIF, respectively, within 60 minutes, with a significantly slowed degradation rate. These data fully demonstrate that the COS coating can provide an effective barrier for GDH-gD-Fc-CSS-BBV in an in vitro simulated gastrointestinal environment, significantly reducing the destruction of the antigen by gastric acid, pepsin, and pancreatic enzymes, thereby ensuring that the vaccine components maintain immune activity before reaching the intestinal mucosal absorption site.

[0088] 5. GDH-gD-Fc-CSS-BBV@COS can be internalized by macrophages.

[0089] To investigate whether GDH-gD-Fc-CSS-BBV@COS, which binds to two antigens, affects cell internalization, RAW264.7 cell line was used for validation. Cytotoxicity was first assessed before validation. The results showed that within a concentration range of 12 mg / mL, GDH-gD-Fc-CSS-BBV@COS did not produce significant cytotoxicity in RAW264.7 cells. Figure 13 Subsequently, the uptake of GDH-gD-Fc-CSS-BBV@COS by RAW264.7 was observed using laser confocal microscopy. The results showed that RAW264.7 could uptake chitosan-encapsulated vesicles regardless of whether the antigen was conjugated. Figure 14 ).

[0090] 6. Oral immunization of GDH-gD-Fc-CSS-BBV@COS mice can reduce the inflammatory response induced by muscle immunity.

[0091] To investigate the in vivo endotoxin activity of GDH-gD-Fc-CSS-BBV@COS, changes in body temperature and serum pro-inflammatory cytokine levels in mice were monitored at 1, 3, 6, and 12 h post-immunization. The results showed that mouse body temperature initially increased and then decreased within 12 hpi. Figure 15 Compared to injection, oral immunization resulted in a smaller increase in body temperature in mice. Serum levels of pro-inflammatory cytokines initially increased, then decreased, peaking at 6 hours. Figure 16Notably, in any CSS-BBV-containing immunization group, the secretion rates of TNF-α and IL-6 were lower than in other groups. Furthermore, serum TNF-α and IL-6 levels in orally administered GDH-gD-Fc-CSS-BBV@COS mice were lower than in the intramuscularly injected group, indicating that oral immunization of GDH-gD-Fc-CSS-BBV@COS mice can reduce the stress response induced by muscle immunity.

[0092] 7. Oral administration of CCS-OMV-GDH-gD-Fc@COS induces high levels of specific IgG antibodies.

[0093] To verify whether the oral nanovesicle vaccine CCS-OMV-GDH-gD-Fc@COS described in this invention can induce a specific humoral immune response, serum was collected from experimental mice via the retroorbital venous plexus at 0, 7, 14, 21, 28, and 35 days post-immunization. The levels of IgG antibodies against GDH, gD, lipopolysaccharide (LPS), and outer membrane proteins (OMPs) were quantitatively detected using indirect ELISA (iELISA). The results are summarized in […]. Figure 17The experimental results showed that at 35 dpi, except for the PBS blank control group, the oral GDH-SpyTag group, and the oral GDH+gD-Fc+BBV+COS unconjugated mixture group, all other treatment groups induced mice to produce high levels of GDH-specific IgG. Among them, the oral CCS-OMV-GDH-gD-Fc@COS group and the intramuscularly injected CCS-OMV-GDH-gD-Fc@COS group showed no statistically significant difference in GDH-specific IgG levels, and both were significantly higher than the commercially available inactivated streptococcal vaccine group. Similarly, in the IgG detection of gD at 35 dpi, except for the PBS blank control group, the oral gD-Fc group alone, and the oral GDH+gD-Fc+BBV+COS unconjugated mixture group, all other groups showed high titers of gD-specific IgG. Among them, the intramuscular injection CCS-OMV-GDH-gD-Fc@COS group induced the highest level of gD-specific IgG, which was significantly higher than that of the commercially available porcine pseudorabies virus inactivated vaccine group. Although the oral CCS-OMV-GDH-gD-Fc@COS group was significantly lower than that of the commercially available inactivated vaccine group, there was no statistically significant difference compared with the intramuscular injection gD-Fc+206 adjuvant positive control group, indicating that the oral route can also produce a high level of gD-specific antibodies. Furthermore, in all immunization groups containing CSS-BBV, the levels of specific IgG antibodies against LPS and OMPs were significantly increased, and the levels in the GDH-gD-Fc-CSS-BBV@COS conjugate group were significantly higher than those in the GDH+gD-Fc+BBV+COS unconjugate mixture group. In summary, the oral CCS-OMV-GDH-gD-Fc@COS nanovesicle vaccine of this invention can induce high-level, multi-target specific IgG antibody responses in mice.

[0094] 8. Oral administration of CCS-OMV-GDH-gD-Fc@COS induces high levels of sIgA antibodies.

[0095] To further evaluate the mucosal immune response induced by the oral nanovesicle vaccine GDH-gD-Fc-CSS-BBV@COS of this invention, vaginal secretions were collected from experimental mice 35 days post-immunization. The levels of specific secretory IgA (sIgA) targeting GDH, gD, LPS, and OMPs were quantitatively detected using iELISA. The results are shown below. Figure 18Experimental results showed that all mice orally administered the CSS-BBV formulation exhibited significantly elevated levels of specific sIgA against LPS and OMPs in their secretions. Importantly, only the group orally administered GDH-gD-Fc-CSS-BBV@COS induced high levels of both GDH and gD-specific sIgA, with statistically significant differences compared to the other groups. These results fully demonstrate that the GDH-gD-Fc-CSS-BBV@COS oral nanovesicle vaccine described in this invention can efficiently deliver antigens to the intestinal mucosa and elicit a significant specific sIgA mucosal immune response.

[0096] 9. Oral administration of CCS-OMV-GDH-gD-Fc@COS induces high levels of PRV neutralizing antibodies.

[0097] Neutralizing antibody titer is a key indicator for evaluating the protective efficacy of PRV vaccine. Serum from mice in each group was collected at 35 days post-contraction (dpi), and the titer of anti-PRV neutralizing antibodies was determined using a neutralization assay. Figure 19 The results showed that the geometric mean titer of neutralizing antibodies in the intramuscularly injected GDH-gD-Fc-CSS-BBV@COS group reached 2. 7.14 Compared with commercial PRV inactivated vaccine group (2 7.53 The levels were at the same level, and significantly higher than the intramuscular injection group of gD-Fc+206 adjuvant (2). 5.87 ) and intramuscular injection of GDH+gD-Fc+BBV+COS unconjugated mixture group (2 6.09 More importantly, mice orally administered GDH-gD-Fc-CSS-BBV@COS produced neutralizing antibody titers of 2. 6.32 There was no significant difference compared to the intramuscular injection group of gD-Fc+206 adjuvant, suggesting that the oral formulation of the present invention can induce neutralizing antibody levels comparable to the injectable formulation under needle-free conditions, and has excellent humoral immune activation ability.

[0098] 10. CCS-OMV-GDH-gD-Fc@COS provides full attack protection against S. suis 2 and PRV.

[0099] To systematically evaluate the in vivo protective effect of GDH-gD-Fc-CSS-BBV@COS on mice, a 10 LD50 assay was performed at 35 dpi. 50 PRV or 50 LD 50Mice in each group were challenged with a lethal dose of Streptococcus suis and observed for 14 consecutive days. Clinical symptom scores showed that, except for deceased individuals, surviving mice in each group experienced a typical "mild-severe-recovery" course of disease during the observation period. Mice in the intramuscular injection and oral administration of GDH-gD-Fc-CSS-BBV@COS group exhibited the mildest clinical symptoms, with scores consistently lower than other treatment groups. Figure 20 Survival rate statistics showed that in mice challenged with PRV, the survival rate was 100% in both the intramuscular injection and oral administration of GDH-gD-Fc-CSS-BBV@COS groups; the survival rate was 83.3% in the intramuscular injection of gD-Fc+206 adjuvant group and the commercially available PRV inactivated vaccine group; and the survival rate of the remaining groups was ≤16.6%. In mice challenged with Streptococcus suis, 100% protection was achieved in the intramuscular injection of the commercially available Streptococcus suis inactivated vaccine group, the intramuscular injection of the unconjugated mixture of GDH+gD-Fc+BBV+COS group, and the intramuscular injection and oral administration of GDH-gD-Fc-CSS-BBV@COS group; the survival rate was 83.3% in the intramuscular injection of GDH+206 adjuvant group; and the survival rate was ≤16.6% in the PBS group, the oral GDH group, and the oral GDH+gD-Fc+BBV+COS unconjugated mixture group. Figure 21 In summary, regardless of whether administered via intramuscular injection or orally, GDH-gD-Fc-CSS-BBV@COS provides 100% immune protection against lethal doses of PRV and Streptococcus suis, significantly outperforming existing commercial vaccines and conventional adjuvant controls. This fully demonstrates that the nanovesicle vaccine of this invention possesses the three major advantages of broad-spectrum, high efficiency, and oral administration, and has outstanding prospects for industrial application.

[0100] Table 3. Immunization doses and routes for each group

[0101]

Claims

1. A method for preparing biomimetic vesicles of Salmonella choleraesuis for oral delivery of dual antigens, characterized in that, It is done in the following steps: Step 1: Clone ClyA, SpyCatcher, and 6×His into the pCold I linearized vector to obtain the pCold I-ClyA-SpyCatcher-6×His plasmid; clone ClyA, SpG, and 6×His into the pCold I linearized vector to obtain the pCold I-ClyA-SpG-6×His plasmid. Step 2: Co-transform pCold I-ClyA-SpyCatcher-6×His plasmid and pCold I-ClyA-SpG-6×His plasmid into SC-L3 competent cells to obtain SC-L3-ClyA-SpyCatcher / SpG recombinant bacteria; The SC-L3 strain was obtained by modifying the SC014 strain. 1) Using the genomic sequence of strain SC014 as a template, PCR amplification was performed using DN-1F / DN-1R and DN-2F / DN-2R primers to obtain upstream and downstream fragments of the tolB, msbB and pagP genes, which were named N-up and N-down, respectively; N in N-up and N-down represents the tolB, msbB and pagP genes, respectively. The DN-1F primers are: DtolB-1F:5'-ttctagtacgtagacacgaatggtcagccatcgaaaga-3' DmsbB-1F:5'-ttctagtacgtagacaccgcagcggcacggcctatctg-3' DpagP-1F: 5'-ttctagtacgtagacactatcccttaatgatgtagcgc-3' The DN-1R primers are: DtolB-1R:5'-ttaattattacatatctcccatacctgggc-3' DmsbB-1R:5'-ctggaaaagcctagtcctgatataggttgac-3' DpagP-1R: 5'-cccttccccgaccgttcaaaaattcgactg-3' The DN-2F primers are: DtolB-2F:5'-ggggagatatgtaataattaattgattacta-3' DmsbB-2F:5'-tcaggactaggcttttccagggtctgctgac-3' DpagP-2F: 5'-tttgaacggtcggggaagggcattgttcag-3' The DN-2R primers are: DtolB-2R: 5'-ggTAATACGTAGACACTacgcctttaccctgcaggta-3' DmsbB-2R:5'-ggTAATACGTAGACACTatggcgaggtcgtggtcgct-3' DpagP-2R: 5'-ggTAATACGTAGACACTaacccgccgaaagggcgggtt-3'; The SC014 strain is Salmonella choleraesuis, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 33887 and deposit date of April 28, 2025. 2) Using 112-F / 112-R primers, PCR amplification was performed with suicide plasmid pRE112 as a template to obtain a linear suicide plasmid vector fragment, which was purified and recovered and named 112-V; then N-up and N-down were ligated to 112-V, and after correct sequencing, the plasmids were extracted to obtain suicide plasmids P1, P2 and P3, respectively. The 112-F primer is 5'-AGTGTCTACGTATTAccgggaattcatgcag-3'; the 112-R primer is 5'-gtgtctacgtactagaagaagcttg-3'; 3) Using primers DpagP-P2-F / R and DmsbB-P3-F / R, linear fragments of P2 and P3 were amplified from the constructed P2 suicide plasmid and P3 suicide plasmid, respectively. Primers DlpxE-F / R and DpagL-F / R were used to amplify the lpxE and pagL fragments from plasmid pQK0055 containing lpxE and pagL fragments. The lpxE fragment was ligated to the P2 linear fragment to obtain the P4 plasmid. The pagL fragment was ligated to the P3 linear fragment to obtain the P5 plasmid. The DpagP-P2-F primer is 5'-cggggaagggcattgttcag-3'; The DpagP-P2-R primer is 5'-accgttcaaaaattcgactgtg-3'; The DmsbB-P3-F primer is 5'-gcttttccagggtctgctgacgc-3'; The DmsbB-P3-R primer is 5'-ctagtcctgatataggttgacag-3'; The DlpxE-F primer is 5'-GCACGCCGTTAACCCGAGA-3'; The DlpxE-R primer is 5'-AGCCACAAATGCTGCTGTGTGG-3'; The DpagL-F primer is 5'-agcttccccggataaccagaagcaataaaaaaagc-3'; The DpagL-R primer is 5'-atcctgttgtgctaaataatctcacgattgc-3'; 4) P1, P4, and P5 plasmids were transformed into competent cells, and after culture, the bacterial solutions were added to LB plates for culture. After chloramphenicol resistance screening, strains with recombinant P1, P4, and P5 plasmid DNA were obtained. After being plated on LB plates containing 10% sucrose and cultured, single colonies were picked and screened again on LB plates containing chloramphenicol resistance. Colony PCR was performed using primers DtolB-1F / DtolB-2R. Those that were positive were the SCΔtolBΔpagPΔmsbB::lpxE::pagL mutant, named SC-L3. Step 3: After expanding the SC-L-CSS recombinant bacteria, IPTG was added for induction, followed by Na2EDTA. The culture was continued, centrifuged, the precipitate was collected, washed, and the bacteria were driven to bud through the gap using an ultra-high pressure homogenizer. The sample passing through the gap was collected, centrifuged, the supernatant was collected, filtered, and CSS-BBV was obtained by molecular sieve chromatography. Step 4: Conjugate CSS-BBV with an antigen fused with SpyTag and Fc to obtain CSS-BBV conjugated with dual antigens fused with SpyTag and Fc. Step 5: Add the chitosan oligosaccharide solution dropwise to the CSS-BBV coupled with SpyTag and Fc dual antigens. After standing, remove the excess chitosan oligosaccharide to obtain the chitosan oligosaccharide-coated CSS-BBV loaded with dual antigens.

2. The method for preparing biomimetic vesicles of Salmonella choleraesuis for oral delivery of dual antigens according to claim 1, characterized in that, The method for preparing SC-L3 competent cells is as follows: 1) Recover SC-L3 from a -80℃ freezer and incubate overnight in a medium containing Amp antibiotic. The next day, transfer the bacterial culture to LB liquid medium and incubate until the OD600 of the bacterial culture is 0.3~0.

7. 2) Centrifuge the bacterial culture at 4°C, discard the supernatant and collect the precipitate; 3) Add pre-cooled 0.1 M CaCl2 solution to the collected precipitate, incubate on ice, centrifuge, and collect the precipitate; 4) Repeat step 3) once; 5) Add pre-cooled 0.1 M CaCl2 solution containing 10% glycerol, mix well, and store at -80℃.

3. The method for preparing biomimetic vesicles of Salmonella choleraesuis for oral delivery of dual antigens according to claim 1, characterized in that, The IPTG induction was performed by adding IPTG to a final concentration of 0.1 mM and inducing culture at 13-17 ℃ for 5-8 h.

4. The method for preparing biomimetic vesicles of Salmonella choleraesuis for oral delivery of dual antigens according to claim 1, characterized in that, The addition of Na2EDTA refers to adding 1~3mM Na2EDTA and continuing to incubate for 2 hours.

5. The method for preparing biomimetic vesicles of Salmonella choleraesuis for oral delivery of dual antigens according to claim 1, characterized in that, The washing described in step three is performed using PBS buffer.

6. The method for preparing biomimetic vesicles of Salmonella choleraesuis for oral delivery of dual antigens according to claim 1, characterized in that, The conditions for the ultra-high pressure homogenizer to drive bacteria to bud through the gaps in step three are 1000~1500 bar, 2~4 times, 4℃.

7. The method for preparing biomimetic vesicles of Salmonella choleraesuis for oral delivery of dual antigens according to claim 1, characterized in that, Step four involves the conjugation of CSS-BBV with the antigen fused with SpyTag and Fc, specifically as follows: First, the CSS-BBV and the antigen of the fusion SpyTag were mixed and stirred in a buffer at 4°C for 1 to 1.5 hours. Then, the uncoupled antigen of the fusion SpyTag was removed using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa to obtain the CSS-BBV with the fusion SpyTag. Next, the CSS-BBV conjugated with SpyTag and the antigen conjugated with Fc were mixed and stirred in the phase buffer at 4°C for 1 to 1.5 hours. Then, the unconjugated antigen conjugated with SpyTag and Fc dual antigen was separated using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa. The buffer solution is a mixture of 20 mM Tris-HCl and 50 mM NaCl, with a pH of 8.

0.

8. The method for preparing biomimetic vesicles of Salmonella choleraesuis for oral delivery of dual antigens according to claim 1, characterized in that, The mass concentration of chitosan oligosaccharide in step five is 2~14 mg / mL, and the pH value is 5.0~6.

0.

9. The method for preparing biomimetic vesicles of Salmonella choleraesuis for oral delivery of dual antigens according to claim 1, characterized in that, The step five involves letting the food stand at 4°C for 30-60 minutes.

10. The biomimetic vesicles of Salmonella choleraesuis prepared according to claim 1 are used to prepare oral delivery dual antigens.

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