Nanometer vaccine vector based on cholesteroid-free capsule protein and application of nanometer vaccine vector

Abstract

The invention provides a nano vaccine vector based on cholesteroid-free capsule protein and application of the nano vaccine vector, and belongs to the technical field of nano particle vaccines. The nano vaccine carrier is formed by sequentially connecting cholesteroid-free capsule protein, extension peptide, linker and molecular glue from an N end to a C end. The invention also provides a nano vaccine based on the cholesteric plasma-free capsule protein. The antigen is obtained by reacting the carrier with an antigen connected with SpyTag, SpyTag002 or SpyTag003. The invention also provides a mycoplasma hyopneumoniae nano vaccine and a new crown nano vaccine. The antigen is obtained by reacting the carrier with an antigen connected with SpyTag003. The nano vaccine vector disclosed by the invention can be used for intramuscular injection vaccines and mucosal vaccines for human and livestock, can remarkably improve the immune effect and enhance the immune response while displaying antigens, has a remarkable self-adjuvant effect, and provides a safe and efficient potential choice for the development of subunit vaccines.

Description

Technical Field

[0001] This invention belongs to the field of nanoparticle vaccine technology, specifically relating to a nanovaccine carrier based on cholesteroid-free capsule protein and its application. Background Technology

[0002] Traditional vaccines consist of complete pathogens, primarily including live attenuated vaccines and inactivated vaccines, which can elicit a strong immune response. However, live attenuated vaccines have drawbacks such as the risk of virulence reversion, significant side effects, and unclear immune mechanisms. Both live attenuated and inactivated vaccines require sophisticated pre-treatment culture processes. Therefore, subunit vaccines with clearly defined immunogenic components have become a hot topic in contemporary vaccine research. However, due to the small size of the antigens in subunit vaccines, highly efficient adjuvants or delivery systems are often needed to enhance their immunogenicity. Studies have shown that the size of the antigen in a subunit vaccine significantly affects the uptake and processing by antigen-presenting cells (APCs). APCs can more effectively process antigens similar in size to the pathogen (20-100 nm). On the other hand, nanoparticles (less than 200 nm) can directly reach lymphatic organs via lymphatic drainage within hours after injection, facilitating antigen transport and processing within the body. Furthermore, high-density repeated display of antigens on the particle surface can promote B cell cross-linking (BCR), eliciting a stronger immune response. Therefore, using nanoparticles as carriers or delivery systems for subunit vaccines is a preferred strategy. Currently, the nanoparticles commonly used as vaccine carriers or delivery systems include Helicobacter pylori ferritin, capsule shell protein / encapsulin, artificial nanoparticles MI3, I53-50AB, and viral capsid particles. However, the self-adjuvant effect of these particles is still insufficient to meet the needs of immune protection. Summary of the Invention

[0003] The purpose of this invention is to provide a nano-vaccine carrier based on cholecystokinin-free capsule protein, which can be used for human and veterinary vaccines. This nano-vaccine carrier can significantly improve the immune effect and enhance the immune response while displaying the antigen, and has a significant self-adjuvant effect.

[0004] This invention also provides a method for preparing a cholesteroid-free capsule protein vaccine vector.

[0005] This invention also provides the application of this cholesteroid-free capsule protein vaccine vector.

[0006] The objective of this invention is achieved through the following technical solution: A nano-vaccine carrier based on cholesteroid-free capsule protein is composed of cholesteroid-free capsule protein, extended peptide, linker and molecular glue linked sequentially from the N-terminus to the C-terminus.

[0007] In this invention, the extended peptide sequence is VLD; the linker is selected from (GSS)n, (GS)n, (GSSGS)n, where n is any natural number greater than or equal to 1; the molecular glue is SpyCatcher, SpyCatcher002, or SpyCatcher003.

[0008] In this invention, the amino acid sequence of the nanovaccine carrier is shown in SEQ ID NO.1.

[0009] The present invention also provides a nanovaccine based on cholesteroid capsule protein, wherein the antigen of the nanovaccine is obtained by reacting the carrier with an antigen linked to SpyTag, SpyTag002 or SpyTag003.

[0010] In this invention, the antigen is a bacterial or viral antigen; the vaccine is an intramuscular vaccine or a mucosal vaccine.

[0011] The present invention also provides a porcine mycoplasma pneumoniae nanovaccine, wherein the antigen of the nanovaccine is obtained by reacting the carrier with porcine mycoplasma pneumoniae antigen linked to SpyTag003.

[0012] In this invention, the porcine mycoplasma pneumoniae antigen linked to SpyTag003 includes P97R1 antigen, P46 antigen and NrdF antigen respectively linked to SpyTag003.

[0013] In this invention, the nanovaccine also contains an adjuvant, such as the oil-in-water adjuvant AddVax.

[0014] The present invention also provides a COVID-19 nanovaccine, wherein the antigen of the nanovaccine is obtained by reacting the carrier with a COVID-19 antigen linked to SpyTag003.

[0015] In this invention, the COVID-19 antigen is the COVID-19 antigen S protein RBD.

[0016] This invention relates to a nano-vaccine carrier based on cholesteroid-free capsule protein modified with molecular glue. By coupling antigens, including single or complex components with immunogenicity relevant to humans and other animals, to the surface of the capsule protein, the constructed capsule protein particles serve as a larger antigen display platform, significantly activating cellular, humoral, and mucosal immunity. Through the spontaneous and rapid formation of covalently bonded peptide-protein reaction pairs between SpyCatcher and SpyTag, SpyCatcher002 and SpyTag002, and SpyCatcher003 and SpyTag003, various antigens can be conveniently displayed on the surface of the particle protein. Therefore, this vaccine carrier based on cholesteroid-free capsule protein provides a safe and efficient potential option for the development of subunit vaccines. Attached Figure Description

[0017] Figure 1 These are the identification results of the recombinant capsule protein Ab-SC. Figure A: SDS-PAGE electrophoresis images of Ab-SC expression identification: 1. Uninduced whole cells; 2. Induced whole cells; 3. Supernatant after disruption; 4. Precipitate after disruption. Figure B: SDS-PAGE electrophoresis images of Ab-SC purification identification: 1. Recombinant capsule protein Ab-SC before purification; 2. Recombinant capsule protein Ab-SC after purification. Figure C: Particle size detection results of recombinant capsule protein Ab-SC; the horizontal axis is diameter in nm; the vertical axis is intensity in %. Figure D: Transmission electron microscopy image of Ab-SC. Figure E shows the detection results of recombinant capsule protein Ab-SC using a HiPrep 16 / 60 Sephacryl S-500 HR spectrometer; the horizontal axis is elution volume in mL; the vertical axis is absorbance at 280 nm.

[0018] Figure 2 These are SDS-PAGE electrophoresis images of each protein before and after purification.

[0019] Figure 3 These are SDS-PAGE electrophoresis images of the recombinant protein Tag-RBD before and after purification. Lanes 1 and 2 are the Tag-RBD before and after purification, respectively.

[0020] Figure 4 The relative mRNA levels of IFN-β and IL-6 in mouse DC cells after stimulation with different protein particles are shown on the ordinate, representing the relative mRNA levels of IFN-β and IL-6, respectively. In this invention, ns indicates P > 0.05; * indicates P ≤ 0.05; ** indicates P ≤ 0.01; *** indicates P ≤ 0.001; **** indicates P ≤ 0.0001.

[0021] Figure 5 Serum IgG and sIgA levels in mice immunized with different particles of OVA antigen are shown. The vertical axis represents IgG titer and OD450nm of sIgA detected in bronchoalveolar lavage fluid, respectively. PBS is the blank control group.

[0022] Figure 6 A schematic diagram of the assembly of Mycoplasma hyopneumoniae intramuscular granule vaccine (MhpAg-Ab), where Ab-P97R1 / P46 / NrdF refers to MhpAg-Ab.

[0023] Figure 7 SDS-PAGE electrophoresis images of MhpAg-Ab before and after purification.

[0024] Figure 8Serum IgG antibody levels in piglets after immunization in each group.

[0025] Figure 9 Results of serum antibody persistence testing after immunization in piglets of each immunization group.

[0026] Figure 10 The viral load in the lung tissue of piglets in each group was detected by qPCR, where the blank group refers to the blank control group. The horizontal axis represents the group.

[0027] Figure 11 The levels of IgG antibodies produced by piglets in each group after immunization with Mycoplasma hyopneumoniae mucosal vaccine.

[0028] Figure 12 Antibody duration of IgG levels in piglets after immunization with Mycoplasma hyopneumoniae mucosal vaccine in each group.

[0029] Figure 13 The results show the viral load in the lung tissue of piglets in each group as detected by qPCR, with the horizontal axis representing the group.

[0030] Figure 14 A schematic diagram illustrating the reaction between the COVID-19 S protein RBD and Ab-SC.

[0031] Figure 15 SDS-PAGE electrophoresis images of Ab-SC particles (RBD-Ab) loaded with SARS-CoV-2 antigen Tag-RBD before and after purification.

[0032] Figure 16 This study demonstrates the antibody IgG levels of intramuscularly injected vaccines containing different nanoparticles of the COVID-19 RBD antigen.

[0033] Figure 17 Antibody duration assay of a nano-intramuscular vaccine (RBD-Ab) displaying the COVID-19 RBD antigen.

[0034] Figure 18 Serum and mucosal antibody levels of different nanoparticle mucosal vaccines.

[0035] Figure 19 Serum antibody duration assay for nano-mucosal vaccine (RBD-Ab). Detailed Implementation

[0036] CHAPS: 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt.

[0037] Example 1: Preparation of Cholesterol-Free Capsule Protein and Antigen Protein 1. Design of a nanovaccine carrier based on cholesteroid-free capsule protein To obtain nanovaccine carriers that can display antigens and significantly improve immunization efficacy and enhance immune responses, numerous nanovaccine carriers were designed and tested. It was found that only the nanovaccine carrier based on cholesteroid-free capsule protein (Genebank accession number TVP97064.1) achieved the desired effect. This cholesteroid-free capsule protein-based nanovaccine carrier is denoted as capsule protein Ab-SC. From the N-terminus to the C-terminus, it consists of cholesteroid-free capsule protein, extended tripeptide (VLD), linker (GSSGSSGSS sequence), and molecular glue SpyCatcher003 linked sequentially. Its amino acid sequence is shown in SEQ ID NO.1. After codon optimization, the coding gene sequence of this capsule protein Ab-SC was obtained, as shown in SEQ ID NO.2.

[0038] The sequence shown in SEQ ID NO.2 was synthesized by GenScript and inserted into the pET-28a(+) vector. Bam HⅠ and Hin Between the dⅢ restriction sites, the expression plasmid pET-28a(+)-Ab-SC was obtained.

[0039] 2. Expression and purification of capsule protein Ab-SC The capsule protein Ab-SC expression plasmid pET-28a(+)-Ab-SC was transformed into competent cells of *E. coli* BL21(DE3) strain using a 42°C heat shock method. The cells were then plated onto LB agar plates containing ampicillin and incubated overnight at 37°C with the plates inverted. Single colonies were picked and cultured in LB liquid medium containing ampicillin at 37°C with shaking for approximately 10 hours. The bacterial culture was then expanded at a 1:100 volume ratio and cultured at 37°C with shaking until the OD reached [value missing]. 600 When the concentration reaches 0.7, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.4 mM, and continue culturing at 18°C ​​for 20 h. Collect the Escherichia coli culture, centrifuge at 6000×g for 20 min, and collect the bacterial precipitate.

[0040] The bacterial pellet was resuspended in purification buffer (pH 8.0, 20 mM Tris-HCl buffer containing 150 mM NaCl, 5 mM β-mercaptoethanol, and 1 mM CHAPS). The resuspended bacterial solution was then sonicated for 60 min at 450 W for 3 seconds followed by a 6-second pause. The sonicated bacterial solution was centrifuged at 50,000 × g for 30 min at 4–8 °C. The supernatant was collected and filtered twice through a 0.22 μm filter. The recombinant capsule protein Ab-SC was purified using a nickel column according to standard methods, eluting the target protein with purification buffer containing 500 mM imidazole. The eluted target protein was dialyzed overnight with purification buffer and then purified using a Capto Core 400 column (purchased from Cytiva) according to the manufacturer's instructions. During purification, this column adsorbs contaminants smaller than 400 kDa; therefore, the recombinant capsule protein Ab-SC (molecular weight 2580 kDa) flowed out with the flow-through. The flow-through was collected and concentrated using an ultrafiltration tube. The concentrated crude protein was purified using a HiPrep 16 / 60 Sephacryl S-500 HR gel filtration chromatography column (purchased from Cytiva). Elution was performed with purification buffer, and the fraction exhibiting a UV absorption peak at 280 nm was collected; this was the target protein, the purified recombinant capsule protein Ab-SC. Figure 1 As shown in Figure A, the recombinant capsule protein Ab-SC was expressed in a soluble manner. The purified recombinant capsule protein Ab-SC was detected by SDS-PAGE electrophoresis, and the results are as follows. Figure 1 B shows a specific band at 43 kDa, as expected, with a purity exceeding 90%. Figure 1 As can be seen from D, Ab-SC can autonomously assemble into spherical nanoparticles. The particle size of the purified recombinant capsule protein Ab-SC was measured using a nanoparticle size analyzer, and the particle size was found to be approximately 35 nm. Figure 1 C), therefore, it can be determined that the expressed recombinant capsule protein Ab-SC self-assembled into polymers, appearing as granules. This was confirmed by the HiPrep 16 / 60 SephacrylS-500 HR assay results ( Figure 1 E) It was confirmed that the polymer has a molecular weight of 2580 kDa and is a hexadecimer.

[0041] 3. Expression and purification of antigen proteins There are three Mycoplasma hyopneumoniae antigens: amino acids 809-893 of the P97R1 antigen (GenBank accession number: WP_016340189.1), amino acids 36-419 of the P46 antigen (GenBank accession number: WP_094521730.1), and amino acids 25-362 of the NrdF antigen (GenBank accession number: WP_220200531.1). The COVID-19 antigen is amino acids 5-227 of the S protein RBD (receptor-binding region) (GenBank accession number: WLQ25843.1). The standard antigen is amino acids 2-386 of ovalbumin OVA (GenBank accession number: AUD54526).

[0042] Three antigen genes of *Mycoplasma hyopneumoniae* were tandemly linked with the SpyTag003 gene (Gene Bank accession number: WWV57716.1, amino acids 111-126), and then optimized according to the codon bias of *E. coli* to obtain Tag-P97R1, Tag-P46, and Tag-NrdF, respectively. The gene encoding the SARS-CoV-2 antigen S protein RBD was tandemly linked with the SpyTag003 gene and optimized according to the codon bias of insect cells to obtain Tag-RBD. The standard antigen OVA was tandemly linked with SpyTag003 and then optimized according to the codon bias of *E. coli* to obtain Tag-OVA. The gene and amino acid sequences of each fusion protein are shown in Table 1.

[0043] Table 1. Gene and amino acid sequences of each fusion protein

[0044] The Tag-P97R1 gene sequence was inserted between the BamI and XhoI restriction sites of pET-32a, and the Tag-P46 and Tag-NrdF gene sequences were inserted between the NdeI and XhoI restriction sites of pET-21a, respectively. Subsequently, each recombinant expression vector was transformed into *E. coli* BL21(DE3) competent cells to construct engineered bacteria expressing the three antigens. Each engineered bacterium was inoculated into LB medium and cultured at 37°C with shaking until OD... 600 The concentration was 0.7, and IPTG was added to a final concentration of 0.4 mM. Expression was induced at 18℃ for 16 hours. The induced engineered bacteria were collected by centrifugation at 4℃, and the cells were lysed by sonication at 4℃. After centrifugation at 15000×g for 30 mins, the supernatant of the cell lysate was collected and filtered twice through a 0.22 μm filter membrane (the resulting protein is referred to as the unpurified protein). The recombinant proteins Tag-P97R1, Tag-P46, and Tag-NrdF were purified using a nickel column. Finally, SDS-PAGE (…) was performed. Figure 2The purity of the purified proteins was assessed. Tag-P97R1, Tag-P46, and Tag-NrdF showed specific bands at 38kDa, 46kDa, and 40kDa, respectively, as expected, indicating a purity of at least 85%.

[0045] The Tag-RBD gene was inserted between the BamH and XhoI restriction sites of the pFastBac1 plasmid, and then transformed into *E. coli* DH10Bac cells. After homologous recombination, positive recombinant rod-like particles were selected by blue-white screening, extracted, and transfected into sf9 insect cells to obtain a recombinant virus expressing the recombinant protein Tag-RBD. This recombinant virus was amplified three times and then transfected into Hi5 cells for high-level expression. Sixty hours after transfection, the supernatant from the insect cells was collected, centrifuged at 15000×g for 30 mins, and filtered through a 0.22 μm filter (the resulting protein was designated as the unpurified protein). The recombinant protein Tag-RBD was purified using a nickel column. Finally, the purity of the purified protein was assessed by SDS-PAGE electrophoresis. Figure 3 As can be seen, a specific band appeared at 29.4 kDa, which is as expected, and the purity reached 90%.

[0046] The standard antigen Tag-OVA gene sequence was inserted between the NdeI and XhoI restriction sites of the expression vector pCold I, and then transformed into *E. coli* BL21(DE3) competent cells to construct an engineered bacterium. The recombinant protein Tag-OVA was expressed and purified according to the expression and purification methods for *Mycoplasma hyopneumoniae* antigen. SDS-PAGE electrophoresis confirmed the presence of a specific band at 46 Da, as expected, indicating a purity of over 85%.

[0047] Example 2: Evaluation of the immunogenicity of different particulate carriers using OVA antigen. To compare the immunomodulatory effects of cholecystokinin-free capsule proteins with other ferritins and capsule protein carriers, dendritic cell stimulation assays were performed using Helicobacter pylori ferritin Hp (gene bank accession number AOO95266.1, amino acids 371-548), Thermocystis aquatica capsule protein Tm (gene bank accession number WP_004080898.1, amino acids 1-265), super-stable MI3 particles (gene bank accession number QNS17493.1, amino acids 130-341), and cholecystokinin-free capsule protein Ab, respectively, to detect the differences in the cellular immune activation effects of these granular proteins. Since these particles will be used for antigen display later, all granular proteins used here were conjugated to SpyCatcher003. The above proteins were tandemly linked with SpyCatcher003 to obtain fusion proteins Hp-SC (corresponding to Helicobacter pylori ferritin Hp), Tm-SC (corresponding to Thermocystis aeruginosa capsule protein Tm), and MI3-SC (corresponding to the superstable particle MI3), respectively, with sequences shown in Table 1. These particles were prepared according to the construction method of the Ab-SC expression plasmid and the expression and purification method of the capsule protein Ab-SC in Example 1. Identification confirmed successful expression, and the particles could spontaneously assemble into multimers, forming nanoscale particles.

[0048] DC cell stimulation test 1.5×10 6 Mouse dendritic cells (DCs) were seeded at 500 μL / well in 24-well plates, with Hp-SC, Tm-SC, MI3-SC, Ab-SC, and LPS intervention groups established (LPS is lipopolysaccharide of Gram-negative bacteria, an activator of TLR4, purchased from Beyotime, product code S1732-5mg). Each group was performed in triplicate, and a blank control group was also included. 10 μL of a solution containing 20 μg of the corresponding antigen (PBS buffer, pH 7.4) was added to each well in each group, while an equal volume of PBS buffer (pH 7.4) was added to each well in the blank control group. Cells were cultured at 37°C and 5% CO2 for 24 hours, and total mRNA was extracted from each well. The relative mRNA levels of IFN-β and IL-6 in each well were determined by quantitative real-time PCR (the detection method is disclosed in Ding X, Sun M, Guo F, et al. Picrasidine S Induces cGAS-Mediated Cellular Immune Response as a Novel Vaccine Adjuvant J. Advanced Science, 2024, 11(32). DOI:10.1002 / advs.202310108.). The results are as follows: Figure 4Cells incubated with Ab-SC particles showed significantly higher IFN-β expression levels compared to other groups. IFN-β is a type I interferon, and its upregulation indicates activation of the cGAS-IFN-I pathway. Therefore, vaccines using Ab as nanocarriers can stimulate T-cell immunity, which is more conducive to enhancing the body's cellular immunity. Cells incubated with Ab-SC particles also showed significantly lower IL-6 secretion levels than the LPS group, indicating that Ab does not induce an excessive inflammatory response and is a safe vaccine carrier.

[0049] 2. Mouse Immunization Experiment To compare the activating effects of the above particles on humoral and mucosal immunity in animals.

[0050] At an antigen-to-carrier molar ratio of 1:1, recombinant protein Tag-OVA (prepared in Example 1) was mixed with Hp-SC, Tm-SC, MI3-SC, and Ab-SC, respectively, and incubated at 4°C for 8 hours for molecular glue coupling reaction. After the reaction, the mixture was purified using a HiPrep 16 / 60 Sephacryl S-500 HR gel filtration chromatography column (purchased from Cytiva) to remove free recombinant protein Tag-OVA, yielding purified OVA-loaded particles HP-OVA (corresponding to Hp-SC), Tm-OVA (corresponding to Tm-SC), MI3-OVA (corresponding to MI3-SC), and Ab-OVA (corresponding to Ab-SC). SDS-PAGE electrophoresis showed a purity of over 85%. Mice were immunized with HP-OVA, Tm-OVA, MI3-OVA and Ab-OVA respectively, and the secretion of serum antibody IgG and mucosal antibody IgA was observed to compare the activation effects of different particles on humoral immunity and mucosal immunity.

[0051] (1) Test vaccines: The purified HP-OVA was diluted to 89.3 μg / ml, Tm-OVA to 97.8 μg / ml, MI3-OVA to 88.2 μg / ml, and Ab-OVA to 96.9 μg / ml with 0.01M PBS buffer at pH 7.4. These were used as test vaccines, and the content of Tag-OVA antigen in each test vaccine was 50 μg / ml. (2) Antigen control vaccine (OVA): Tag-OVA was diluted to 50 μg / ml with 0.01M PBS buffer at pH 7.4.

[0052] (4) Immunization test in mice Female BALB / c mice were randomly divided into six groups: HP-OVA experimental vaccine group, Tm-OVA experimental vaccine group, MI3-OVA experimental vaccine group, Ab-OVA experimental vaccine group, antigen control vaccine (OVA) group, and blank control group (immunized with 0.01M PBS buffer at pH 7.4). Five mice were in each group for the immunization experiment. The first immunization was performed on day 0, with each group receiving the corresponding vaccine via intranasal administration at a dose of 100 μL per mouse. A second immunization was performed on day 14 after the first immunization, using the same method and dose. On day 28 after the initial immunization, serum and bronchoalveolar lavage fluid were collected from each group of mice. Serum IgG levels and mucosal antibody IgA levels were measured (Feng Zhi-xin, Shao Guo-qing, Liu Mao-jun, et al. Development and validation of a SIgA-ELISA for the detection of Mycoplasma hypopneumoniae infection [J]. Vet Microbiol, 2010, 143 (2-4): 410-416.). Results are as follows: Figure 5 When the same antigen OVA was displayed on different particle surfaces, differences were observed in serum antibody IgG levels and mucosal antibody IgA (sIgA) levels. For serum antibodies, there was no significant difference between the antigen control vaccine (OVA) group and the blank control group, but the granulated OVA group showed a significant increase in serum antibody titers compared to the antigen control vaccine (OVA) group. The Ab-OVA experimental vaccine group had the highest antibody titer, reaching 1:51200, which was nearly ten times higher than the Tm-OVA experimental vaccine group (1:6400), which also contained a capsule protein. Furthermore, the antibody level in the Ab-OVA experimental vaccine group was four times that of the HP-OVA experimental vaccine group (1:12500). Similarly, the mucosal antibody IgA (i.e., sIgA) detection results also showed that the Ab-OVA-stimulated mucosal antibody level was significantly higher than other groups (the antibody titer in the Ab-OVA experimental vaccine group was 1.8 times that of the HP-OVA experimental vaccine group). Therefore, among the many particulate carriers currently available, cholesteroid-free capsule protein has significant advantages as a mucosal vaccine carrier, as it can more strongly stimulate the body's humoral and mucosal immunity, and stimulate the production of higher levels of serum IgG antibodies and mucosal IgA antibodies.

[0053] Example 3: Immunization efficiency and challenge protection test of Mycoplasma hyopneumoniae intramuscular injection vaccine To investigate the immune enhancement effect of the capsule protein Ab-SC prepared in Example 1 on the antigen via intramuscular injection, an intramuscular vaccine and a control vaccine based on Ab-SC were prepared, and immune efficiency and challenge protection tests were conducted on piglets.

[0054] 1. Preparation of various vaccines (1) Swine Mycoplasma pneumoniae intramuscular vaccine: showing swine Mycoplasma pneumoniae triple antigen vaccine (MhpAg-Ab) Tag-P97R1, Tag-P46, and Tag-NrdF were mixed in a molar ratio of 1:1:1. To ensure sufficient antigen display on the particle surface, the mixture was incubated overnight at 4°C to obtain a mixture of three antigens. This mixture of three antigens was then mixed with Ab-SC in a molar ratio of 6:1 (the molar ratio for the three antigen mixtures refers to the total molar number of the three antigens). An isopeptide coupling reaction was then performed between SpyCatcher003 and SpyTag003 at 4°C. Figure 6 The reaction time was 8 hours. After the reaction, molecular sieve chromatography using HiPrep 16 / 60 Sephacryl S-500 HR (Cytiva) was performed according to standard methods to remove free Tag-P97R1, Tag-P46, and Tag-NrdF, yielding purified Ab-SC particles simultaneously loaded with P97R1, P46, and NrdF, named MhpAg-Ab. Figure 7 It can be seen that the purity of the purified MhpAg-Ab reaches over 85%.

[0055] MhpAg-Ab standard dose vaccine: The purified MhpAg-Ab was diluted to 250 μg / ml with 0.01M PBS buffer (pH 7.4), with a total content of 200 μg / ml for Tag-P97R1, Tag-P46, and Tag-NrdF. The 250 μg / ml MhpAg-Ab solution was mixed with the oil-in-water adjuvant AddVax (InvivoGen, USA) at a volume ratio of 1:1 to obtain the MhpAg-Ab standard dose vaccine, denoted as MhpAg-Ab.

[0056] Low-dose MhpAg-Ab vaccine: Purified MhpAg-Ab was diluted to 50 μg / ml with 0.01M PBS buffer (pH 7.4), containing a total content of 40 μg / ml of Tag-P97R1, Tag-P46, and Tag-NrdF. The 50 μg / ml MhpAg-Ab solution was mixed with the oil-in-water adjuvant AddVax (InvivoGen, USA) at a volume ratio of 1:1 to obtain the low-dose MhpAg-Ab vaccine, denoted as MhpAg-AbL.

[0057] (2) Antigen control vaccine MhpAg standard dose vaccine: Tag-P97R1, Tag-P46 and Tag-NrdF were mixed in a molar ratio of 1:1:1 and diluted with 0.01M PBS buffer at pH 7.4 to a total concentration of 200 μg / ml. Then, it was mixed with the oil-in-water adjuvant AddVax (InvivoGen, USA) in a volume ratio of 1:1 to obtain the MhpAg standard dose vaccine, denoted as MhpAg.

[0058] MhpAg low-dose vaccine: The three proteins Tag-P97R1, Tag-P46 and Tag-NrdF were mixed in a molar ratio of 1:1:1 and diluted with 0.01M PBS buffer at pH 7.4 to a total concentration of 40 μg / ml. Then, it was mixed with the oil-in-water adjuvant AddVax (InvivoGen, USA) in a volume ratio of 1:1 to obtain the MhpAg low-dose vaccine, denoted as MhpAgL.

[0059] (3) Vector control vaccine Ab-SC was diluted to 200 μg / ml with 0.01M PBS buffer at pH 7.4, and mixed with oil-in-water adjuvant AddVax (InvivoGen, USA) at a volume ratio of 1:1 to obtain the vector control vaccine, denoted as Ab.

[0060] (4) Blank control vaccine: PBS buffer with pH 7.4 and a concentration of 0.01M.

[0061] (5) MhpAg-Hp vaccine: MhpAg-Hp was prepared according to the preparation method of MhpAg-Ab, except that Hp-SC was used instead of Ab-SC to perform isopeptide coupling reaction with Tag-P97R1, Tag-P46 and Tag-NrdF. The purified MhpAg-Hp was diluted with 0.01M PBS buffer at pH 7.4 to a total content of 200 μg / ml of Tag-P97R1, Tag-P46 and Tag-NrdF, and then mixed with the oil-in-water adjuvant AddVax (InvivoGen, USA) at a volume ratio of 1:1 to obtain the MhpAg-Hp vaccine.

[0062] The preparation methods for the above-mentioned antigens and carriers are described in Example 1. The grouping and antigen content of the above-mentioned vaccines are shown in Table 2.

[0063] Table 2. Immunization Efficiency Test of Mycoplasma hyopneumoniae Intramuscular Vaccine in Swine and Grouping of Animals for Challenge Protection

[0064] 2. Animal grouping and immunization program Forty-two three-way crossbred piglets that were negative for common swine pathogens (African swine fever virus, porcine respiratory and reproductive syndrome virus, porcine circovirus, porcine infectious diarrhea virus, pseudorabies virus, Streptococcus suis, Haemophilus parasuis, Mycoplasma hyopneumoniae, and Mycoplasma hyopneumoniae) and had not been vaccinated were randomly divided into groups 1-10. The group names and vaccines administered are shown in Table 2. Groups 1-6 each contained 5 pigs, and groups 7-10 each contained 3 pigs. On day 0 (D0) and day 14 (D14), each pig was injected with 1 ml of the corresponding vaccine or 1 ml of PBS buffer into the right triangular neck muscle.

[0065] 3. Calculation of virus challenge and virus protection rate Six weeks after the initial immunization, groups 1-6 (immunization challenge group, vector control group, and challenge control group) were challenged with Mycoplasma hyopneumoniae. Group 7 (blank control group) received the same dose of PBS buffer via the same route. The specific challenge method was as follows: challenge was administered via tracheal injection, with a dose of 5 ml per head, for a total challenge volume of 10... 9 .5 The *Mycoplasma hyopneumoniae* strain AV747 (disclosed in ZL201410287236.8) from the CCU was challenged 24 hours later, followed by a second challenge using the same strain, method, and dosage. The specific method for tracheal injection was as follows: The pig was fixed in a supine position with its head down using a slanted restraint frame. *Mycoplasma hyopneumoniae* strain AV747 was injected via the trachea. The pig must remain still and immobilized for at least 5 seconds after inoculation. Four weeks after the first challenge (i.e., 10 weeks after the initial immunization), necropsy was performed on the immune challenge group, challenge control group, vector control group, and blank control group. A 28-point scoring method was used to calculate the degree of lung lesions and the challenge protection rate.

[0066] Statistics and evaluation of protection rate after challenge: Ten weeks after the first immunization, experimental pigs were necropsized, and lung lesions in each group were scored according to the 28-point scoring method [Madec F, Kobish M (1982) Gross lung lesions of pigs at slaughter. Journéesde la Recherche Porcine 14:405–412 (in French)]. The lung scoring method was as follows: 0.5 for lesion percentage of 0–12.5%; 1 for lesion percentage of 12.6–25%; 1.5 for lesion percentage of 25.1–37.5%; 2 for lesion percentage of 37.6–50%; 2.5 for lesion percentage of 50.1–62.5%; 3 for lesion percentage of 62.6–75%; 3.5 for lesion percentage of 75.1–87.5%; and 4 for lesion percentage of 87.6–100%. The pig lung is divided into seven lobes: left apical lobe, left cardiac lobe, left diaphragmatic lobe, right apical lobe, right cardiac lobe, right diaphragmatic lobe, and accessory lobe. Based on the percentage of lesions in each lobe, the lung lesion index for the entire lung of each pig was calculated (published in Maddec F KM: Bilan lésionnel des poumons de porcs charcutiers àl'abattoir. Journées de la Recherche Porcine en France (in French) 1982, 14:8.). The average lung lesion index of pigs in each experimental group was calculated. The reduction rate of the lung lesion index was calculated using the following formula: Lung lesion index reduction rate = (average lung lesion index of the challenged control group - average lung lesion index of the immunized challenge group) / (average lung lesion index of the challenged control group - average lung lesion index of the blank control group) × 100%. Finally, the vaccine efficacy was calculated according to the formula in the third edition of "Principles of Epidemiology in Public Health Practice" published by the US CDC: Vaccine efficacy = (Number of cases in the non-immunized group - Number of cases in the immunized challenge group) / Number of cases in the non-immunized group, where the non-immunized group corresponds to the challenge control group in the animal grouping.

[0067] Table 3. Statistics on the efficacy rate and reduction rate of lung lesion index of various swine mycoplasma pneumoniae intramuscular injection vaccines.

[0068] Note: In Table 3, the comma-separated numbers in the lung lesion index represent the lung lesion index of one piglet. For example, "0, 4, 0, 0, 0" means that the lung lesion indices of 5 piglets are 0, 4, 0, 0, 0 respectively.

[0069] The results (Table 3) show that the vaccine efficacy rate in the MhpAg-Ab immunization challenge group (received with the standard dose of vaccine) was 80%, and the lung lesion index reduction rate was 92.5%; the vaccine efficacy rate in the MhpAg-AbL immunization challenge group (received with the low dose of vaccine) was 80%, and the lung lesion index reduction rate was 86.8%; the vaccine efficacy rate in the MhpAg vaccine immunization challenge group (received with the standard dose of vaccine) was 20%, and the lung lesion index reduction rate was 64.2%; the vaccine efficacy rate in the MhpAgL immunization challenge group (received with the low dose of vaccine) was 0%, and the lung lesion index reduction rate was 32.1%; and the vaccine efficacy rate in the vector control group was 0%, and the lung lesion index reduction rate was 3.8%. These results indicate that the vaccine containing cholesteroid-free capsule protein at the same dose provides significantly higher immunoprotective effects in animals than the antigen control vaccine. Furthermore, when MhpAg-Ab is used to immunize animals at a low dose, its immunoprotective effect can reach a level comparable to the standard dose, demonstrating that cholesteroid-free capsule protein has significant advantages as a vaccine carrier.

[0070] 4. Enzyme-linked immunosorbent assay (ELISA) to detect serum IgG levels. Blood samples were collected from pigs in the immunized challenge group, challenge control group, vector control group, and blank control group for serum separation. Blood collection points were: before the first immunization (D0), before the second immunization (D14), 28 days after the first immunization (D28), and before challenge (D42). Blood samples were collected from immunized pigs every 3 months until they reached 24 months of age for serum separation. Serum IgG levels were detected using a porcine mycoplasma pneumoniae antibody detection kit (purchased from IDEXX, USA, product number 99-06733). The detection was performed according to the kit instructions, and the S / P ratio (S is the absorbance value of the sample, P is the absorbance value of the positive control) was calculated. A positive result was defined as S / P ≥ 0.4.

[0071] The results are as follows Figure 8As shown, both the standard-dose and low-dose MhpAg-Ab vaccines produced high levels of IgG, with little difference between the two. The standard-dose MhpAg-Ab vaccine produced the highest antibody levels, reaching an S / P of 2.38 on day 28, and maintaining a high level thereafter until day 70. The low-dose MhpAg-Ab vaccine produced an S / P of 2.04 on day 28, and maintained a high level thereafter until day 70. The antibody levels produced by both the standard-dose and low-dose MhpAg vaccines were significantly lower than those of the standard-dose and low-dose MhpAg-Ab vaccines. In the group receiving the standard-dose MhpAg vaccine, the S / P reached 1.17 on day 28 and remained at this level until day 70; in the group receiving the low-dose MhpAg vaccine, the S / P reached 0.77 on day 28 and remained at this level until day 70. The antibody levels were lowest in the vector control group, the challenge control group, and the blank control group, with little difference among them, basically at the baseline level.

[0072] From the results of the duration of immunity ( Figure 9 Based on the results, the standard dose of MhpAg-Ab vaccine stimulates high-titer antibodies in pigs that can last for at least 2 years. The antibody immunity produced by the MhpAg-Hp vaccine lasts for 270 days (after which the S / P ratio is less than 0.4), while the antibody immunity produced by the standard dose of MhpAg vaccine lasts only 180 days, after which the S / P ratio is less than 0.4. Therefore, both antibody levels and duration indicate that the particulate carrier has a longer antibody duration, and the cholesteroid-free capsule protein significantly and effectively enhances the strength and duration of humoral immunity compared to Helicobacter pylori ferritin.

[0073] 5. Peripheral blood mononuclear cell (PBMC) proliferation detection The study examined the specific proliferation of peripheral blood mononuclear cells in experimental pigs after immunization with different vaccines, following stimulation by Mhp antigen, to evaluate the level of specific cellular immune response.

[0074] Peripheral blood was collected from each immune challenge group, vector control group, and blank control group on day 42 (42 days after the first immunization). Porcine peripheral blood mononuclear cells were isolated from each group using a porcine peripheral blood mononuclear cell separation kit (Beijing Solarbio Science & Technology Co., Ltd., P4420). 90 μl of cells containing 1 × 10¹² cells were seeded into each well of a 96-well cell plate. 5Peripheral blood mononuclear cells were cultured in 1640 medium (containing 10% fetal bovine serum). Then, 3 μg of a 1:1:1 mixture of P97R1, P46, and NrdF proteins (10 μL) was added to each well for PBMC stimulation. Positive and negative control wells were also included in each cell group. Positive control wells were stimulated with 3 μg of concanavalin A instead of antigen, while negative control wells were stimulated with the same volume of PBS buffer. Blank control wells were also included, containing only 1640 medium containing 10% fetal bovine serum. After incubating the 96-well cell plate at 37°C in a 5% CO2 incubator for 72 hours, 10 μL of CCK-8 solution (Beyotime) was added to each well, and incubation continued for another 4 hours. The OD values ​​of each well were then read using a microplate reader. 450 The cell stimulation index is calculated as follows: SI = (antigen stimulation well OD) / (OD value of cell stimulation well). 450 Value - Blank control well OD 450 (value) / (OD of negative control well) 450 Value - Blank control well OD 450 value) The results, as shown in Table 4, indicate that, except for the vector control group (Ab), all other immunization challenge groups showed enhanced immune response and peripheral blood mononuclear cell proliferation after Mhp antigen stimulation. Specifically, the MhpAg-Ab or MhpAg-AbL immunization challenge groups containing the particulate vaccine showed significantly higher stimulation indices, more than 2.5 times higher than the groups receiving the simple antigen vaccine. The MhpAg-Ab immunization challenge group receiving the standard dose of the MhpAg-Ab vaccine showed slightly higher indices, but the difference was not statistically significant. The stimulation indices of the MhpAg and MhpAgL immunization challenge groups receiving the simple antigen vaccine were significantly lower than those of the particulate vaccine groups, with the low-dose group showing even lower stimulation indices. Therefore, it can be seen that vaccines prepared using cholecystokinin-free capsule protein as a carrier have a significant ability to stimulate cellular immune responses.

[0075] Table 4. Stimulation index (SI) and specific IL-17 secretion of mononuclear cells isolated from each vaccine group after antigen stimulation.

[0076] 6. Detection of IL-17 secretion by peripheral blood mononuclear cells The ELISA method was used to detect the induction and secretion of specific IL-17 in peripheral blood mononuclear cells of experimental pigs after stimulation with Mhp antigen.

[0077] Peripheral blood was collected from each immune challenge group, vector control group, and blank control group at day 42. Porcine peripheral blood mononuclear cells were isolated from each group using a porcine peripheral blood mononuclear cell separation kit (Beijing Solarbio Science & Technology Co., Ltd., P4420). In 96-well cell culture plates, 90 μL of cells containing 1 × 10⁶ cells were seeded into each well. 5 Peripheral blood mononuclear cells were cultured in 1640 medium (containing 10% fetal bovine serum). Then, 3 μg of a 1:1:1 mixture of P97R1, P46, and NrdF proteins was added for PBMC stimulation (10 μL volume). Positive and negative control wells were set up for each cell group. Positive control wells were stimulated with 3 μg of concanavalin A instead of antigen, while negative control wells were stimulated with the same volume of PBS buffer. After incubation at 37°C and 5% CO2 for 72 hours, the cell culture supernatant from each well was aspirated, and the IL-17 content in each well was measured using a porcine interleukin-17A detection kit (Beijing Solarbio Science & Technology Co., Ltd., SEKP-0173).

[0078] The results are shown in Table 4. Except for the vector control group, all other immune challenge groups stimulated the release of IL-17 from peripheral blood mononuclear cells after Mhp antigen stimulation. The MhpAg-Ab and MhpAg-AbL immune challenge groups showed high secretion levels, with the MhpAg-Ab group showing a slightly higher level, but the difference was not statistically significant. The synthesis and secretion of IL-17 in the MhpAg and MhpAgL immune challenge groups were significantly lower than those in the MhpAg-Ab group, and the low-dose group showed even lower IL-17 secretion. Therefore, it can be seen that the vaccine prepared using cholecystokinin-free capsule protein as a carrier has a significant ability to induce IL-17 secretion.

[0079] 7. Detection of pathogen load in porcine tissues using qPCR method Ten weeks after the first immunization (i.e., 28 days after challenge), bronchoalveolar lavage fluid (BALF) was isolated from the experimental pigs. If there was any damage to the lungs, the damaged area should be clamped with hemostatic forceps first. Then, 50 mL of autoclaved PBS buffer (0.01 M, pH=7.4) was drawn into the lungs through a syringe and injected along the trachea. Each lung lobe was gently massaged by hand. After massaging for 2 minutes, the fluid in the lungs was poured out, which is the bronchoalveolar lavage fluid. 1 mL of BALF was drawn and centrifuged at 4℃ and 10000×g for 20 mins. The precipitate was collected, and DNA was extracted from the precipitate using a nucleic acid extraction kit. The pathogen nucleic acid content in the immune challenge group, challenge control group, vector control group, and blank control group was detected by qPCR (for specific methods, see Wu Yuzi et al., Establishment and application of TaqMan-BHQ fluorescence quantitative PCR detection method for Mycoplasma hyopneumoniae P97, Chinese Journal of Veterinary Science, 2012, 42(12): 1268-1272).

[0080] result( Figure 10 The results showed that the nucleic acid content of *Mycoplasma hyopneumoniae* in the bronchoalveolar lavage fluid of each immunized challenge group was lower than that of the challenge control group and the vector control group to varying degrees. The nucleic acid content in the bronchoalveolar lavage fluid of the MhpAg-Ab and MhpAg-AbL immunized challenge groups was the lowest, with no significant difference between them and between them and the blank control group. The nucleic acid content of the MhpAg-Ab (10 copies) and MhpAg-AbL (15 copies) immunized challenge groups was also significantly lower than that of the MhpAg (2500 copies) and MhpAgL (50000 copies) groups. The nucleic acid content of the MhpAg immunized challenge group was significantly lower than that of the challenge control group (251188 copies) and the vector control group (125892 copies), but the nucleic acid content of the MhpAgL immunized challenge group was not significantly different from that of the challenge control group and the vector control group. Therefore, it can be seen that the *Mycoplasma hyopneumoniae* intramuscular injection vaccine prepared using cholesteroid-free capsule protein as a carrier has a significant protective effect against challenge.

[0081] Example 4: Challenge protection test of porcine Mycoplasma pneumoniae mucosal vaccine based on cholesteroid capsule protein. To investigate the immune enhancement effect of the capsule protein Ab-SC prepared in Example 1 on the antigen via nasal drop, the immunization efficiency and challenge protection of piglets were tested using the Ab-SC-based porcine mycoplasma pneumoniae mucosal vaccine and the control vaccine.

[0082] 1. Preparation of Mycoplasma hyopneumoniae mucosal vaccine and control vaccine MhpAg-Ab standard dose vaccine: The purified MhpAg-Ab was diluted to 125 μg / ml with 0.01M PBS buffer at pH 7.4. The total content of Tag-P97R1, Tag-P46 and Tag-NrdF was 100 μg / ml, which yielded the MhpAg-Ab standard dose vaccine, denoted as MhpAg-Ab.

[0083] Low-dose MhpAg-Ab vaccine: The purified MhpAg-Ab was diluted to 25 μg / ml with 0.01M PBS buffer at pH 7.4. The total content of Tag-P97R1, Tag-P46 and Tag-NrdF was 20 μg / ml, and the resulting low-dose MhpAg-Ab vaccine was denoted as MhpAg-AbL.

[0084] MhpAg standard dose vaccine: Mix the three proteins Tag-P97R1, Tag-P46 and Tag-NrdF in a molar ratio of 1:1:1, and dilute with 0.01M PBS buffer at pH 7.4 to a total concentration of 100 μg / ml to obtain the MhpAg standard dose vaccine, denoted as MhpAg.

[0085] MhpAg low-dose vaccine: Mix the three proteins Tag-P97R1, Tag-P46 and Tag-NrdF in a molar ratio of 1:1:1, and dilute with 0.01M PBS buffer at pH 7.4 to a total concentration of 20 μg / ml to obtain the MhpAg low-dose vaccine, denoted as MhpAgL.

[0086] Vector control vaccine: Dilute Ab-SC with 0.01M PBS buffer (pH 7.4) to 100μg / ml to obtain the vector control vaccine, denoted as Ab.

[0087] Blank control group: pH 7.4, 0.01M PBS buffer.

[0088] MhpAg-Hp vaccine: The purified MhpAg-Hp was diluted with 0.01M PBS buffer at pH 7.4 to a total concentration of Tag-P97R1, Tag-P46 and Tag-NrdF of 100 μg / ml to obtain the MhpAg-Hp vaccine.

[0089] 2. Animal grouping and immunization program Forty-two three-way crossbred piglets that were negative for common swine pathogens (African swine fever virus, porcine respiratory and reproductive syndrome virus, porcine circovirus, porcine infectious diarrhea virus, pseudorabies virus, Streptococcus suis, Haemophilus parasuis, Mycoplasma hyopneumoniae, and Mycoplasma hyopneumoniae) and had not been vaccinated were randomly divided into groups 1-10. The group names and vaccines administered are shown in Table 5. Groups 1-6 each contained 5 pigs, and groups 7-10 each contained 3 pigs. The corresponding vaccines were administered intranasally or with 1 ml of PBS on day 0 (D0) and day 14 (D14).

[0090] Table 5. Animal groups in the immunogenicity test and challenge protection test of various porcine Mycoplasma hyopneumoniae mucosal vaccines.

[0091] 3. Calculation of virus challenge and virus protection rate Six weeks after the initial immunization, groups 1-6 (immunization challenge group, vector control group, and challenge control group) were challenged with Mycoplasma hyopneumoniae. Group 7 (blank control group) was injected with the same dose of PBS buffer via the same route. Four weeks after the initial challenge (i.e., 10 weeks after the initial immunization), necropsy was performed on the immunization challenge group, challenge control group, and blank control group. The severity of lung lesions was calculated using a 28-point scoring method, and the protection rate against the virus was calculated.

[0092] The virus strains used for challenge, the challenge methods, the challenge dosage, the lung score, the calculation of the lung lesion index, the reduction rate of the lung lesion index, and the calculation of the vaccine efficacy were the same as in Example 3.

[0093] Table 6. Statistics on efficacy and reduction rate of lung lesion index of porcine mycoplasma pneumoniae nanocarrier mucosal vaccine.

[0094] The results (Table 6) showed that the vaccine efficacy rate in the MhpAg-Ab immunization challenge group was 80%, and the lung lesion index reduction rate was 90.6%; the vaccine efficacy rate in the MhpAg-AbL immunization challenge group was 80%, and the lung lesion index reduction rate was 88.7%; the vaccine efficacy rate in the MhpAg immunization challenge group was 0%, and the lung lesion index reduction rate was 20.8%; the vaccine efficacy rate in the MhpAgL immunization challenge group was 0%, and the lung lesion index reduction rate was 18.9%; and the vaccine efficacy rate in the vector control group was 0%, and the lung lesion index reduction rate was 7.5%. These results indicate that the Ab-SC-based porcine mycoplasma pneumoniae mucosal vaccine at the same dose provided significantly better immunoprotective effects than the antigen control vaccine. Furthermore, when animals were immunized with a low dose of the MhpAg-Ab vaccine, its immunoprotective effect was comparable to that of the standard dose, demonstrating the significant advantages of cholesteroid-free capsule protein as a mucosal vaccine carrier.

[0095] 4. Enzyme-linked immunosorbent assay (ELISA) to detect serum IgG levels. Blood samples were collected from pigs in the immunized challenge group, challenge control group, vector control group, and blank control group for serum separation. Blood collection time points were: before the first immunization (D0), before the second immunization (D14), 28 days after the first immunization (D28), and before challenge (D42). Blood samples were collected from immunized pigs every 3 months until they reached 24 months of age. Serum IgG levels were detected using a Mycoplasma pneumoniae antibody detection kit (purchased from IDEXX, USA, product number 99-06733). The S / P ratio was calculated, and a positive result was defined as S / P ≥ 0.4.

[0096] The results are as follows Figure 11As shown, both the standard-dose and low-dose MhpAg-Ab vaccines produced high levels of IgG, with no significant difference between them. On day 28, the antibody S / P values ​​reached 2.08 and 1.70, respectively, and remained at high levels until day 70. The antibody levels produced by both the standard-dose and low-dose MhpAg vaccines were very low, similar to the antibody levels in the vector control group, challenge control group, and blank control group, essentially at baseline. On day 28 post-vaccination, the antibody S / P values ​​for the standard-dose and low-dose MhpAg vaccines reached 0.77 and 0.57, respectively. Therefore, cholecystokinin-free capsule protein administered via nasal drops can significantly stimulate the production of serum antibodies.

[0097] From the duration of immunity ( Figure 12 The results showed that the standard dose of MhpAg-Ab vaccine stimulated high-titer antibodies in pigs for at least 2 years. The antibody duration of the MhpAg-Hp vaccine was significantly shorter than that of MhpAg-Ab, but slightly longer than that of the MhpAg vaccine. On day 180 post-vaccination, the S / P ratio of the MhpAg vaccine was <0.4, therefore, the antibody duration was 90 days. On day 360 post-vaccination, the S / P ratio of the MhpAg-Hp vaccine was <0.4, and the antibody turned negative, therefore, the antibody duration of the MhpAg-Hp vaccine was 270 days. The standard dose of the MhpAg-Ab vaccine maintained a high antibody level for 720 days. Therefore, it can be seen that after intranasal immunization, the cholesteroid-free capsule protein significantly enhanced the strength and duration of serum IgG antibodies compared to the simple antigen and Helicobacter pylori ferritin carrier.

[0098] 5. Peripheral blood mononuclear cell (PBMC) proliferation detection The study examined the specific proliferation of peripheral blood mononuclear cells in experimental pigs after immunization with different vaccines, following stimulation by Mhp antigen, to evaluate the level of specific cellular immune response.

[0099] Peripheral blood was collected from each immune challenge group, vector control group, and blank control group at day 42. Porcine peripheral blood mononuclear cells were isolated from each group using a porcine peripheral blood mononuclear cell separation kit (Beijing Solarbio Science & Technology Co., Ltd., P4420). 90 μL of cells containing 1 × 10¹² cells were seeded into each well of a 96-well cell plate. 5Peripheral blood mononuclear cells were cultured in 1640 medium (containing 10% fetal bovine serum). Subsequently, 3 μg of a 1:1:1 mixture of P97R1, P46, and NrdF proteins was added to each well for PBMC stimulation (10 μL volume). Positive and negative control wells were also included in each cell group. Positive control wells were stimulated with 3 μg of concanavalin A instead of antigen, while negative control wells were stimulated with the same volume of PBS buffer. Blank control wells contained only 90 μL of 1640 medium containing 10% fetal bovine serum. After incubating the 96-well cell plate at 37°C in a 5% CO2 incubator for 72 hours, 10 μL of CCK-8 solution was added to each well, and incubation continued for another 4 hours. OD values ​​were then read using a microplate reader. 450 The cell stimulation index is calculated as follows: SI = (antigen stimulation well OD) / (OD value of cell stimulation well). 450 Value - Blank control well OD 450 (value) / (OD of negative control well) 450 Value - Blank control well OD 450 value).

[0100] The results (Table 7) showed that both the standard-dose and low-dose MhpAg-Ab vaccines based on cholecystokinin-free capsule protein had high stimulation indices, with the standard-dose MhpAg-Ab vaccine showing a slightly higher index, but the difference was not statistically significant. In contrast, the stimulation indices of the standard-dose and low-dose MhpAg vaccines containing the simple antigen were very low, similar to those of the vector control group and the blank control group. Therefore, it can be seen that vaccines prepared using cholecystokinin-free capsule protein as a carrier have a significant ability to stimulate cellular immune responses via the mucosal immune pathway.

[0101] Table 7. Stimulation index (SI) of mononuclear cells isolated from piglets in each group after antigen stimulation.

[0102] 6. Detection of IL-17 secretion by peripheral blood mononuclear cells The ELISA method was used to detect the induction and secretion of specific IL-17 in peripheral blood mononuclear cells of experimental pigs after immunization with various vaccines after stimulation by Mhp antigen.

[0103] Peripheral blood was collected from each immune challenge group, vector control group, and blank control group at day 42. Porcine peripheral blood mononuclear cells were isolated from each group using a porcine peripheral blood mononuclear cell separation kit (Beijing Solarbio Science & Technology Co., Ltd., P4420). In 96-well cell culture plates, 1 × 10⁶ cells were separated from each well. 5Peripheral blood mononuclear cells (PBMCs) were seeded at a total volume of 90 μL. Subsequently, a mixture of P97R1, P46, and NrdF proteins at a molar ratio of 1:1:1 was added for PBMC stimulation. The total protein content of the protein mixture was 3 μg, and the volume of the protein mixture was 10 μL. Positive and negative control wells were set up for each cell group. Positive control wells were stimulated with 3 μg of concanavalin A instead of antigen, while negative control wells were stimulated with the same volume of PBS buffer. After incubating the 96-well cell plate at 37°C and 5% CO2 for 72 hours, the supernatant of the cell culture medium was aspirated from each well, and the IL-17 content in each well was detected using a porcine interleukin-17A detection kit (Beijing Solarbio Science & Technology Co., Ltd., SEKP-0173).

[0104] The results are shown in Table 7. Both the standard-dose MhpAg-Ab vaccine and the low-dose MhpAg-Ab vaccine prepared with cholecystokinin-free capsule protein showed high secretion levels, with the MhpAg-Ab immunized challenge group showing slightly higher levels, but the difference was not statistically significant. However, the synthesis and secretion of IL-17 in the MhpAg and MhpAgL immunized challenge groups were significantly lower than those of the standard-dose MhpAg-Ab vaccine, and close to those in the blank control group and the carrier control group, suggesting that simple nasal immunization with protein alone cannot induce a significant mucosal immune response in pigs. Therefore, the mucosal vaccine prepared using cholecystokinin-free capsule protein as a carrier has a significant ability to induce IL-17 secretion.

[0105] 7. Detection of pathogen load in porcine tissues using qPCR method Ten weeks after the first immunization (i.e., 28 days after the challenge), bronchoalveolar lavage fluid (BALF) was isolated from the experimental pigs. If there was any damage to the lungs, the damaged area should be clamped with hemostatic forceps first. Then, 50 mL of autoclaved PBS buffer (0.01 M, pH=7.4) was drawn into the lungs through a syringe and injected along the trachea. Each lung lobe was gently massaged by hand. After massaging for 2 minutes, the fluid in the lungs was poured out, which is the bronchoalveolar lavage fluid. 1 mL of BALF was drawn and centrifuged at 10000×g for 20 mins at 4℃. The precipitate was collected, and DNA was extracted from the precipitate using a nucleic acid extraction kit. The pathogen nucleic acid content in the immune challenge group, challenge control group, vector control group, and blank control group was detected by qPCR (the specific method is the same as that used by Wu Yuzi et al., Establishment and application of quantitative PCR detection method for Mycoplasma hyopneumoniae P97 TaqMan-BHQ, Chinese Journal of Veterinary Science, 2012, 42(12): 1268-1272).

[0106] result( Figure 13The results showed that the nucleic acid content of *Mycoplasma hyopneumoniae* in the bronchoalveolar lavage fluid of the MhpAg-Ab (8.75 copies) and MhpAg-AbL (12.9 copies) immune challenge groups was significantly lower than that of the MhpAg immune challenge group (31,622 copies), the MhpAgL immune challenge group (63,095 copies), the challenge control group, and the vector control group. There was no significant difference between the MhpAg-Ab and MhpAg-AbL immune challenge groups, nor between them and the blank control group. The nucleic acid content of *Mycoplasma hyopneumoniae* in the bronchoalveolar lavage fluid of the MhpAg and MhpAgL immune challenge groups was similar to that of the challenge control group and the vector control group, which was very high. Therefore, the *Mycoplasma hyopneumoniae* mucosal vaccine prepared using cholesteroid-free capsule protein as a carrier has a significant protective effect against viral challenge.

[0107] Example 5: Preparation and mouse immunization test of a novel coronavirus intramuscular vaccine based on cholesteroid capsule protein. To investigate whether the immune-enhancing effect of the capsule protein Ab-SC is applicable to viral proteins, and to evaluate the advantages of the capsule protein Ab-SC as a vaccine carrier, different types of vaccines against the COVID-19 S protein RBD were prepared and subjected to immunization experiments in mice.

[0108] Various types of vaccines involved in the preparation trials The COVID-19 antigen is amino acids 5-227 of the S protein RBD (receptor-binding region) (GeneBank accession number: WLQ25843.1).

[0109] (1) Experimental vaccines: Different nanoparticle vaccines demonstrating the SARS-CoV-2 S protein RBD antigen RBD-Ab: Tag-RBD and Ab-SC prepared in Example 1 were coupled with SpyCatcher003 and SpyTag003 at a molar ratio of 3:1 at 4°C using an isopeptide coupling reaction. Figure 14 The reaction time was 8 hours. Then, following standard methods, free Tag-RBD was removed by HiPrep 16 / 60 Sephacryl S-500 HR (Cytiva) molecular sieve chromatography to obtain purified Tag-RBD-loaded Ab-SC particles, named RBD-Ab. Figure 15 It can be seen that the purity of the purified RBD-Ab reaches over 85%.

[0110] The purified RBD-Ab was diluted to a concentration of 230 μg / ml with 0.01 M PBS buffer (pH 7.4), containing 100 μg / ml of antigen Tag-RBD. The 230 μg / ml RBD-Ab solution was then mixed with the oil-in-water adjuvant AddVax (InvivoGen, USA) at a volume ratio of 1:1 to obtain the RBD-Ab vaccine.

[0111] RBD-Hp, RBD-Tm, and RBD-MI3 were obtained according to the preparation and purification method of RBD-Ab, with the only difference being that Hp-SC (corresponding product RBD-Hp), Tm-SC (corresponding product RBD-Tm), and MI3-SC (corresponding product RBD-MI3) prepared in Example 2 were used to replace Ab-SC in the isopeptide coupling reaction with Tag-RBD. The concentrations of RBD-Hp, RBD-Tm, and RBD-MI3 solutions were 227 μg / ml, 269 μg / ml, and 238 μg / ml, respectively, and the content of the antigen protein Tag-RBD was 100 μg / ml for all of them. RBD-Hp, RBD-Tm, and RBD-MI3 at concentrations of 227 μg / ml, 269 μg / ml, and 238 μg / ml, respectively, were mixed with the oil-in-water adjuvant AddVax (InvivoGen, USA) at a volume ratio of 1:1 to obtain RBD-Hp, RBD-Tm, and RBD-MI3 vaccines.

[0112] (2) Antigen control vaccine (RBD): Tag-RBD (prepared in Example 1) was diluted to a concentration of 100 μg / ml with pH 7.4, 0.01M PBS buffer, and mixed with oil-in-water adjuvant AddVax (InvivoGen, USA) at a volume ratio of 1:1 to obtain the antigen control vaccine (RBD).

[0113] (3) Vector control vaccine (Ab): Dilute Ab-SC (prepared in Example 1) with PBS buffer at pH 7.4 and 0.01M to a concentration of 100 μg / ml, and mix it with oil-in-water adjuvant AddVax (InvivoGen, USA) at a volume ratio of 1:1 to obtain the vector control vaccine (Ab).

[0114] Blank control vaccine (PBS): pH 7.4, 0.01M PBS buffer.

[0115] 2. Mouse immunization experiments and antibody persistence monitoring Female BALB / c mice were randomly divided into seven groups: RBD-Ab vaccine group, RBD-Hp vaccine group, RBD-Tm vaccine group, RBD-MI3 vaccine group, antigen control vaccine group (RBD group), vector control vaccine group (Ab group), and blank control vaccine group (PBS group), with 5 mice in each group. Immunization experiments were conducted. Mice were first immunized on day 0 by injecting the corresponding vaccine (100 μL / mouse) into the gastrocnemius muscle. A second immunization was performed on day 14 after the first immunization, using the same method and dosage. Serum samples were collected from each group on day 28 after the first immunization to detect the level of specific anti-RBD IgG in the serum (the detection method is disclosed in Feng Zhi-xin, Shao Guo-qing, Liu Mao-jun, et al. Development and validation of a SIgA-ELISA for the detection of Mycoplasmahyopneumoniae infection [J]. VetMicrobiol, 2010, 143 (2-4): 410-416.). Figure 16 The results showed that the serum antibody levels in the RBD-Ab, RBD-Hp, RBD-Tm, and RBD-MI3 vaccine groups were significantly higher than those in the antigen control vaccine group, vector control vaccine group, and blank control vaccine group. The antibody levels in the antigen control vaccine group were also significantly higher than those in the vector control vaccine group and the blank control vaccine group. Among the experimental vaccine groups, the serum antibody level in the RBD-Ab vaccine group was significantly higher than that of other types of nanoparticle vaccines. These results indicate that cholesteroid-free capsule proteins, as vaccine carriers, can be applied to viral antigens, strongly stimulating the body's immunity and producing high levels of antibodies; furthermore, among various nanoparticles, cholesteroid-free capsule proteins have a certain advantage in the level of serum antibody production against antigens.

[0116] To monitor the duration of serum IgG antibody production after RBD-Ab immunization in mice, site-directed antibody levels were measured over a period of up to 120 days. Two groups were established: an RBD-Ab vaccine group and an antigen control vaccine (RBD) group, with three female BALB / c mice randomly assigned to each group. The corresponding vaccines were administered via multiple intramuscular injections on day 0 and day 14, with an immunization dose of 100 μL per mouse. Blood samples were collected from the orbital sinus every 10 days, and serum IgG levels were measured using ELISA. Figure 17 The results showed that serum antibody levels in the RBD-Ab vaccine group remained high throughout the 120-day observation period, while the serum antibody duration in the RBD group was only about 60 days, and the antibody levels were lower. Therefore, cholesteroid-free capsule protein is also suitable for viral antigens and has significant advantages as a vaccine carrier in maintaining high antibody levels.

[0117] Example 6: Preparation of a novel coronavirus mucosal vaccine based on cholesteroid capsule protein and mucosal immunization test in mice. To investigate whether cholesteroid-free capsule protein loaded with viral antigens also has the same immune-enhancing effect via the mucosal route, a novel coronavirus mucosal vaccine based on cholesteroid-free capsule protein and novel coronavirus vaccines based on different nanocarriers were prepared and subjected to mouse immunization experiments.

[0118] 1. The preparation methods for the various vaccines involved in the experiment are as follows: (1) Experimental vaccine group: Nanoparticle vaccine displaying the SARS-CoV-2 S protein RBD antigen RBD-Ab, RBD-Hp, RBD-Tm, and RBD-MI3 were prepared and purified using the same method as in Example 5. The purified RBD-Ab, RBD-Hp, RBD-Tm, and RBD-MI3 were diluted to a final Tag-RBD concentration of 50 μg / ml with 0.01 M PBS buffer (pH 7.4) and designated as RBD-Ab, RBD-Hp, RBD-Tm, and RBD-MI3 vaccines, respectively.

[0119] (2) Antigen control vaccine (RBD): Tag-RBD was diluted to 50 μg / ml with 0.01 M PBS buffer at pH 7.4. (3) Vector control vaccine (Ab): Ab-SC was diluted to 50 μg / ml with 0.01 M PBS buffer at pH 7.4. (4) Blank control group: 0.01 M PBS buffer at pH 7.4.

[0120] The methods for preparing the carriers and antigen proteins mentioned above are described in Examples 1 and 2.

[0121] 2. Mouse immunization experiments and antibody persistence monitoring Female BALB / c mice were randomly divided into seven groups: RBD-Ab vaccine group, RBD-Hp vaccine group, RBD-Tm vaccine group, RBD-MI3 vaccine group, antigen control vaccine group (RBD group), vector control vaccine group (Ab group), and blank control group (PBS group), with 5 mice in each group. The mice were first immunized on day 0 by intranasal administration of the corresponding vaccine at a dose of 100 μL per mouse. A second immunization was administered on day 14 after the first immunization, using the same method and dose. Serum and bronchoalveolar lavage fluid were collected from mice in each group on day 28 after the first immunization. Serum IgG and mucosal antibody IgA levels were detected (the detection method is disclosed in Feng Zhi-xin, Shao Guo-qing, Liu Mao-jun, et al. Development and validation of a SIgA-ELISA for the detection of Mycoplasmahyopneumoniae infection [J]. Vet Microbiol, 2010, 143 (2-4): 410-416.). Figure 18 The results showed that serum IgG antibody levels in all experimental vaccine groups were significantly higher than those in the RBD group, the vector control vaccine group, and the blank control group, while there were no significant differences in serum antibody levels among the RBD group, the vector control vaccine group, and the blank control group. Among the different nanoparticle vaccines, the serum antibody level in the RBD-Ab vaccine group was higher than that in other groups. Similarly, for mucosal antibody levels, all experimental vaccine groups were significantly higher than those in the RBD group, the vector control vaccine group, and the blank control group, while there were no significant differences among the RBD group, the vector control vaccine group, and the blank control group. Among the different nanoparticle vaccines, the mucosal antibody level in the RBD-Ab vaccine group was significantly higher than that in other groups. Therefore, these results indicate that cholesteroid-free capsule protein, as a mucosal vaccine carrier, is also suitable for viral antigens and can strongly stimulate humoral and mucosal immunity, producing high levels of serum IgG antibodies and mucosal IgA antibodies.

[0122] To monitor the duration of serum IgG antibody production in mice immunized with the RBD-Ab vaccine, site-directed antibody levels were measured over a period of up to 120 days. Two groups, the RBD-Ab vaccine group and the antigen control vaccine group (RBD), were established, with three female BALB / c mice randomly assigned to each group. The mice were immunized intranasally with the corresponding vaccine (prepared as described in Title 1 of this embodiment) on days 0 and 14, at a dose of 100 μL per mouse. Blood samples were collected from the orbital sinus every 10 days, and serum IgG levels were detected using an ELISA method (the detection method is disclosed in Feng Zhi-xin, Shao Guo-qing, Liu Mao-jun, et al. Development and validation of a SIgA-ELISA for the detection of Mycoplasma hypopneumoniae infection [J]. Vet Microbiol, 2010, 143 (2-4): 410-416.). Figure 19 The results showed that the serum antibody levels in the RBD-Ab vaccine group remained high throughout the 120-day observation period, while the serum antibody duration in the antigen control vaccine group (RBD) was only about 60 days. Therefore, cholesteroid-free capsule protein is also suitable as a mucosal vaccine carrier for viral antigens and has a significant advantage in maintaining high antibody levels.

Claims

1. A nanovaccine carrier based on aacholesteroid capsule protein, characterized in that The amino acid sequence of the nano-vaccine carrier is shown as SEQ ID NO.

1.

2. The nanovaccine vector of claim 1, wherein The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted.

3. The nanovaccine vector according to claim 1 or 2, characterized in that The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted.

4. A nano-vaccine based on aacharidio-protein capsule protein, characterized in that The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted.

5. The nanovaccine according to claim 4 or 5, characterized in that The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted.

6. A Mycoplasma hyopneumoniae nanovaccine, characterized in that The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted.

7. The nanovaccine of claim 6, wherein The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted.

8. The nanovaccine of claim 7, wherein The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted.

9. A COVID-19 nanovaccine characterized in that The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted.

10. The SARS-CoV-2 nanovaccine of claim 9, wherein The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted. The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted. The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted. The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted. The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted. The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted. The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted. The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted. The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted. The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted. The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted. The antigen of the nano-vaccine is obtained after the carrier of claim 1 and the antigen connected with SpyTag, SpyTag002 or SpyTag003 are reacted. The antigen of the nano-v

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