FcRn-targeted Brucella multi-epitope nano vaccine as well as preparation method and application thereof

Abstract

The invention provides an FcRn-targeted Brucella multi-epitope nano vaccine, which can realize long-term protection, shows continuous immune memory, has high-frequency hair-growing central B cells (GCB), follicular helper T cells (TFH) and central memory T cells (TCM), and ensures long-term immune surveillance; even six months after immunization, the serum IgG titer is still obviously higher than that of a control group; efficient antigen delivery can be achieved, specifically, a chitosan-based nano-particle system is adopted, and antigen protection and intestinal epithelium uptake are enhanced through FcRn targeted ligand modification; the delivery efficiency can be improved by 10 times, and the gastrointestinal mucosal barrier is overcome; in addition, rapid immune starting can be achieved, multi-dimensional immune response is induced within 14 days after final immunization, and rapid protection is provided for resisting brucella infection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a chitosan nanovaccine and its preparation method. Background Technology

[0002] Brucellosis is a zoonotic disease with a global impact, causing immense harm and resulting in economic losses exceeding $3 billion annually to the livestock industry, severely hindering its healthy development. Even more serious is the fact that Brucella can also cause severe complications in humans through contact with infected animals or consumption of unsterilized dairy products, such as endocarditis and nerve damage, posing a grave threat to human health.

[0003] Brucella bacteria are relatively stable in the natural environment, surviving in soil and water for weeks or even months, making their spread wider and their control more difficult. Once an outbreak occurs, it not only leads to widespread infection, miscarriage, decreased productivity, and even death in livestock, but also severely damages the entire industry chain, from farming to meat and dairy processing and sales, with economic losses increasing exponentially. For humans, brucellosis infection often results in prolonged physical suffering and economic burden. Symptoms such as recurrent fever, excessive sweating, and fatigue can last for months or even years, severely impacting labor capacity and placing a heavy burden on families and society. Furthermore, brucellosis outbreaks can have a cascading effect on regional economic and social stability, hindering local development and triggering public health panic; its harm is comprehensive and multi-layered.

[0004] Given the serious harm caused by Brucella, vaccine development is particularly necessary. An effective Brucella vaccine is not only an urgent need for controlling epidemics, reducing economic losses, and protecting human health, but also a key measure for maintaining ecological balance and promoting the sustainable development of agriculture and animal husbandry. However, current traditional attenuated live vaccines have many problems. They may lead to infection in immunodeficient hosts or accidental infection in occupationally exposed individuals, with a needlestick injury infection rate of 0.4-6%. Furthermore, they primarily induce systemic immunity and cannot effectively induce mucosal immune responses (sIgA level <1μg / mL), making it difficult to block initial pathogen adhesion. They are also cold-chain dependent, requiring low-temperature storage (2-8℃), limiting their application in livestock fields. Simple multi-epitope vaccines (MEVs) also suffer from low delivery efficiency; free antigens are easily digested by gastrointestinal enzymes (half-life <30 minutes), lack a targeting mechanism, have a mucosal penetration rate of <5%, insufficient immunogenicity, and cannot activate cross-presentation pathways. CD8+ T cells should... The response is weak (activation rate <10%), with poor clearance of intracellular bacteria and short-lived memory maintenance. Germinal center response duration is short (GCB frequency decreases by >80% after 14 days), lacking long-term protection. Conventional mucosal vaccine delivery systems also have problems such as antigen escape, non-specific uptake, and immune tolerance. Unmodified nanoparticles are easily trapped by the mucus layer (permeability <20%), and cannot be efficiently delivered to mucosa-associated lymphoid tissue (MALT). They rely on passive diffusion or macropinocytosis, and the targeting efficiency of antigen-presenting cells (APCs) is low (DC activation rate <15%). Repeated exposure to non-target antigens may induce regulatory T cell (Treg) expansion, weakening protective immunity.

[0005] Against this backdrop, the development of novel vaccines has become crucial to overcoming the challenge of brucellosis control. Currently, the development of novel vaccines presents three major technological breakthroughs. In the field of subunit vaccines, vaccines based on recombinant pathogen proteins have made breakthroughs in biosafety, but the breadth of immune responses induced by single antigens is limited, with animal model protection rates reaching only 65%-78%, indicating significant room for improvement in the comprehensiveness of their immunization effects. In terms of delivery system innovation, the emergence of mRNA complexation technology has brought new ideas to vaccine development. It utilizes the natural targeting of bacterial shell structures to enhance antigen presentation efficiency, while achieving broad-spectrum protection through mRNA encoding multiple epitope antigens. However, it faces the challenge of industrial production costs being 3-5 times higher than traditional vaccines, limiting its large-scale application. Molecular marker vaccines, through gene editing to construct wboA / bp26 double-deleted strains, form molecular identification markers while retaining immunogenicity, solving the problem of distinguishing vaccines from natural infections. However, the insufficient mucosal colonization ability of attenuated strains leads to a shortened duration of immune protection, raising concerns about the long-term protective effect of vaccines. Therefore, there is an urgent need to develop a new targeted delivery Brucella multi-epitope vaccine. Summary of the Invention

[0006] This invention provides a Brucella multi-epitope peptide, a Brucella multi-epitope vaccine containing such a Brucella multi-epitope peptide, and a chitosan Brucella multi-epitope vaccine. It also provides a Brucella multi-epitope vaccine with a novel delivery system that synergistically applies FcRn reverse transport and FcγR cross-presentation to vaccine design.

[0007] Specifically, this invention provides a Brucella multi-epitope peptide, which is composed of the following peptide sequences linked together: amino acid sequences 56-65 of OMP25, amino acid sequences 57-66 of OMP25, amino acid sequences 163-171 of OMP25, amino acid sequences 71-80 of VirB10, amino acid sequences 72-81 of VirB10, amino acid sequences 79-88 of VirB10, amino acid sequences 49-63 of OMP25, amino acid sequences 50-64 of OMP25, amino acid sequences 306-320 of VirB10, amino acid sequences 206-320 of VirB10, amino acid sequences 206-320 of VirB10, amino acid sequences 3 ... The amino acid sequences of positions 65-279, positions 366-380 of VirB, positions 45-60 of OMP25, positions 165-180 of OMP25, positions 182-197 of OMP25, positions 235-250 of VirB10, positions 175-190 of VirB10, positions 309-324 of VirB10, positions 83-98 of VirB10, positions 280-295 of VirB10, and positions 122-239 of VirB10.

[0008] The present invention also provides a Brucella multi-epitope vaccine, which includes the multi-epitope peptide and h-tFc protein described above. The h-tFc protein is added to the N-terminus of the multi-epitope peptide, and the multi-epitope peptide and h-tFc are linked by GGGS to form a recombinant protein.

[0009] The h-tFc in this invention is derived from FcRn. The FcRn targeting backbone is derived from human IgG1 Fc (UniProt ID: P01857), which was selected because of its high affinity for FcγRI and low binding affinity for FcγRIIB. FcRn is encoded by the FCGRT gene and is not only present in the embryonic period, but also widely present in adult parenchymal cells (epithelial, endothelial, hepatocyte and keratinocyte) and hematopoietic cells. FcRn is mainly present on the membranes of endosomes and lysosomes. Structurally, it can form non-covalent binding with β-microglobulin and participate in the circulation between endosomes, lysosomes and cell membranes.

[0010] The h-tFc in this invention is obtained by modifying FcRn, including the following modifications: E318A, K320A, K322A, C226S, C229S, M252Y, S254T, T256E, H433K, and N434F; C1q binding is eliminated by E318A / K320A / K322A mutations to prevent complement activation; monomerization is achieved by C226S / C229S substitution, breaking interchain disulfide bonds to prevent dimer formation; the amino acid sequence is shown in SEQ ID NO.1; the FcRn affinity-enhancing mutations inspired by Efgartigimod (M252Y / S254T / T256E / H433K / N434F) are designed using ABDEG technology to enhance pH-dependent FcRn binding, thereby improving stability under physiological pH gradients and prolonging serum half-life.

[0011] The FcRn targeting mechanism in this invention may be compatible with other intracellular bacteria (such as Salmonella) or viruses (such as HIV mucosal infections).

[0012] Furthermore, the amino acid sequence of the recombinant protein formed by adding h-tFc protein to the N-terminus of the above-mentioned multipeptide site is shown in SEQ ID NO.2.

[0013] The present invention also provides a recombinant nucleic acid molecule that encodes the above-mentioned recombinant protein.

[0014] Furthermore, the sequence of the nucleic acid molecule is shown in SEQ ID NO.3.

[0015] The present invention also provides a chitosan nanovaccine, specifically, a multi-epitope vaccine described above is encapsulated by chitosan.

[0016] The present invention also provides a method for preparing the above-mentioned chitosan nanovaccine, comprising the following steps:

[0017] (1) Dissolve chitosan in acetic acid and filter;

[0018] (2) Mix the chitosan solution obtained in step (1) with the multi-epitope vaccine described above, add sodium tripolyphosphate, and stir to complete gelation.

[0019] The above-mentioned multi-epitope peptides, multi-epitope peptide vaccines, recombinant nucleic acid molecules or chitosan nanovaccines are used in the preparation of drugs for the prevention or improvement of diseases caused by Brucella.

[0020] Furthermore, the above-mentioned drugs are administered orally.

[0021] The present invention has the following beneficial effects:

[0022] I. Reduce the risk of zoonotic diseases

[0023] This vaccine can reduce the risk of zoonotic diseases, especially through mucosal immunity, which can effectively block the transmission of Brucella through the digestive and respiratory tracts, reduce the chance of infection for occupationally exposed groups such as herders and veterinarians, provide strong protection for susceptible populations, and reduce their likelihood of contracting the disease in their daily work.

[0024] II. Unique Targeted Delivery Mechanism

[0025] The vaccine has created a novel targeted delivery mechanism, and for the first time, it has applied the synergistic combination of FcRn reverse transport and FcγR cross-presentation to the design of Brucella vaccines, providing a reliable route for the efficient delivery of Brucella vaccines. It also creates a general technical framework for the development of vaccines against other mucosal pathogens such as influenza virus and Mycobacterium tuberculosis, and promotes the development and progress of the field of mucosal vaccine research and development.

[0026] III. Advantages of Delivery Carriers and Precise Delivery

[0027] The vaccine utilizes chitosan nanoparticles as an oral delivery carrier, combined with FcRn targeting ligand modification to improve antigen stability, protect the antigen from enzymatic degradation, enhance intestinal epithelial cell uptake efficiency, overcome the gastrointestinal mucosal barrier, and achieve precise delivery. This ensures the vaccine exerts its effects more accurately after entering the body, improving the overall efficacy of the vaccine. Furthermore, its delivery system possesses pH responsiveness, enzyme resistance, and enhanced penetration capabilities, enabling it to withstand gastric acid degradation (pH 1.5-3.5), intestinal enzyme hydrolysis, and physical barriers from the mucus layer (pore size <).

[0028] Overcoming challenges such as 200nm, by controlling the nanoparticle size (100-200nm) and surface charge (Zeta potential > +30mV), the specific regional distribution of FcRn in the intestine (mainly in the jejunal epithelium) is precisely located, ensuring the accuracy, effectiveness and stability of vaccine delivery in all aspects.

[0029] IV. Long-lasting immunity and immune memory

[0030] This vaccine achieves a new standard for long-lasting immunity. Through continuous activation of GCB / TCM, it provides protection for over 6 months, overcoming the limitation of traditional mucosal vaccines requiring frequent booster immunizations (every 3-6 months), thus enhancing the convenience and compliance of vaccine use. Simultaneously, the vaccine exhibits sustained immune memory, with a high frequency of germinal center B cells (GCB), follicular helper T cells (TFH), and central memory T cells (TCM) present in the body, ensuring long-term immune surveillance. Even 6 months post-immunization, serum IgG titers remain significantly higher than the control group, maintaining a durable immune defense against Brucella.

[0031] V. Highly efficient antigen delivery and rapid immune initiation

[0032] By utilizing a chitosan-based nanoparticle system combined with FcRn-targeting ligand modification, antigen protection and intestinal epithelial uptake are enhanced, achieving a 10-fold increase in delivery efficiency. This ensures the vaccine reaches its site of action smoothly and efficiently, maximizing its efficacy. Furthermore, the vaccine can induce a multidimensional immune response within 14 days after final immunization, rapidly providing protection against Brucella infection, establishing an immune barrier in a timely manner to resist bacterial invasion, effectively shortening the infection window period, and reducing the risk of infection.

[0033] VI. Convenience of Modular Antigen Design

[0034] The use of modular antigen design (MEV epitope substitution) significantly shortens the research and development cycle, enabling it to be applied more quickly to the emergency prevention and control of zoonotic diseases. In the face of sudden outbreaks, it can provide immune protection more promptly, improving the speed of emergency response. Simultaneously, the multi-epitope antigens in the vaccine balance the spatial conformation of T / B cell epitopes, avoiding epitope masking and maintaining the binding activity of the Fc domain and FcRn. Attached Figure Description

[0035] Figure 1 The target design diagram for h-tFc-MEV;

[0036] Figure 2 The solubility analysis diagram of h-tFc-MEV is shown.

[0037] Figure 3 A graph illustrating the immunization simulation profile of h-tFc-MEV vaccination;

[0038] Figure 4 The structural characterization diagram of the h-tFc-MEV vaccine protein;

[0039] Figure 5 Figure showing the heterologous expression analysis of recombinant MEV and h-tFc-MEV proteins in Escherichia coli;

[0040] Figure 6 Characterization and analysis of the structure and surface charge of h-tFc-MEV and MEV encapsulated by chitosan nanoparticles;

[0041] Figure 7 Characterization analysis diagram of chitosan nanoparticles;

[0042] Figure 8 This is a graph showing the quantitative analysis of serum antigen levels after oral administration;

[0043] Figure 9 A graph showing antigen-specific humoral immunity and Th1 / Th2 characteristics in vaccinated mice;

[0044] Figure 10A graph illustrating the enhanced antigen presentation and immune activation of FcRn-targeted vaccines;

[0045] Figure 11 Flow cytometry analysis of the changes in the ratio of IFN-γ+ and IL-4+CD4 / CD8+ T cells in the spleen of mice 14 days after final immunization;

[0046] Figure 12 CD4 in the spleen + / CD8 + A graph showing the proportion of central memory T cells (TCMs);

[0047] Figure 13 (a) Follicular helper T cell (TFH) subsets in mesenteric lymph nodes (MLN) and (b) spleen;

[0048] Figure 14 A diagram showing the analysis of germinal center B cell (GCB) subsets in MLN and (b) spleen;

[0049] Figure 15 (a) Graph showing the IL-21 level in the spleen as measured by ELISA and (b) the antigen-specific IgG (IgG1 / IgG2a) titer in serum;

[0050] Figure 16 This refers to the mucosal antibody response following h-tFc-MEV vaccination;

[0051] Figure 17 This is a diagram analyzing bacterial colonization in various organs after viral challenge. Detailed Implementation

[0052] The physicochemical analysis process of VirB10 and Omp25 in this invention is as follows: The amino acid sequences of VirB10 and Omp25 were obtained from the UniProt database version 2024_04 (website: https: / / www.uniprot.org), and then their physicochemical properties were analyzed using ProtParam 2.3.1 (website: http: / / web.expasy.org / protparam / ), including molecular weight, theoretical isoelectric point (pI), amino acid / atomic composition, extinction coefficient, predicted half-life, instability index, aliphatic index, and total average hydrophobicity (GRAVY).

[0053] The homology assessment process in this invention is as follows: Sequence homology analysis of the human proteome (classification number: 9606) was performed using NCBI BLASTP 2.14.0+ (website: https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins) with an E value threshold of 0.00130, in order to exclude possible human homologs of natural proteins (VirB10 and Omp25) and vaccine constructs.

[0054] The antigenicity assessment process in this invention is as follows: the antigenicity of candidate proteins and vaccine components is predicted using the VaxiJen 2.0 server (website: http: / / www.ddg-pharmfac.net / vaxijen / VaxiJen / VaxiJen.html), bacterial antigen classification is performed using a species-specific threshold of 0.4, and all assessed peptides meet the minimum length requirement (>5 amino acids).

[0055] The sensitization screening process in this invention is as follows: Based on the machine learning platform AllerTOP v3.0 (website: https: / / www.ddg-pharmfac.net / AllerTOP / N3), potential sensitization is analyzed by amino acid composition and physicochemical properties, and peptides with more than 5 residues are systematically filtered by bioinformatics.

[0056] The transmembrane topology prediction process in this invention is as follows: Transmembrane helices are identified and membrane positioning directions are determined using TMHMM 2.0 (website: https: / / services.healthtech.dtu.dk / service.php?TMHMM-2.0), thereby guiding subsequent epitope screening and excluding hydrophobic transmembrane domains.

[0057] The signal peptide identification process in this invention is as follows: The neural network algorithm of SignalP 5.0 (website: https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0) is used to detect N-terminal signal peptides. Signal peptide regions with a probability > 0.7 are excluded from epitope screening, and the focus is on mature protein domains.

[0058] The toxicity analysis process in this invention is as follows: ToxinPred 1.0 (website: https: / / webs.iiitd.edu.in / raghava / toxinpred / ) is used in combination with alignment and machine learning methods to evaluate candidate epitopes. Non-toxic epitopes with a toxicity score <0.5 are screened and retained for vaccine design using an SVM system.

[0059] The T-cell epitope prediction process in this invention is as follows: Cytotoxic T-cell (CTL) and helper T-cell (HTL) epitopes are identified using the IEDB v3.3 system (https: / / www.iedb.org). CTL prediction employs an artificial neural network (ANN), targeting major HLA class I alleles (HLA-A02:01, HLA-B07:02, HLA-B35:01) with 8-11mer peptides and percentile thresholds <1.0. HTL epitopes are predicted using a combinatorial peptide-MHC class II binding affinity matrix, considering 15mer peptides and HLA-DR variants (DRB101:01, DRB104:01, DRB107:01).

[0060] The B-cell epitope characterization process in this invention is as follows: Linear B-cell epitopes (LBEs) are predicted using a bidirectional recurrent neural network model of ABCpred's RNNv2.1 (https: / / webs.iiitd.edu.in / raghava / abcpred / ) with a 16-mer sliding window analysis (threshold: 0.7 specificity). Conformal B-cell epitopes (CBEs) are mapped using ElliPro's Thornton-antibody method combined with solvent accessibility and salience index calculations. Spatial epitopes are visualized using PyMOL 2.5.8 (accessed April 18, 2024) (minimum salience index 0.6).

[0061] The multi-epitope vaccine construction process in this invention is as follows: To target the Fc receptor, the human IgG1 Fc domain (UniProt P01857) is engineered: C226S / C229S substitution is introduced to eliminate interchain disulfide bonds (monomerization); E318A / K320A / K322A mutations eliminate complement C1q binding; based on the Efgartigimod FcRn affinity optimization paradigm, five pH-sensitive residues (M252Y / S254T / T256E / H433K / N434F) are modified to enhance endosomal circulation. Finally, the engineered human tFc (h-tFc) is fused to the MEV via a GGGS flexible connector to ensure the correct spatial orientation of the antigen module and the Fc effector domain.

[0062] The solubility analysis process in this invention is as follows: The solubility of h-tFc-MEV in the *E. coli* expression system is predicted using the machine learning algorithm of Protein-SOL (https: / / protein-sol.manchester.ac.uk). The model integrates parameters such as charge-hydrophobicity ratio (Z-score < -0.5), aggregation tendency (TANGO score < 5%), and disorder probability (PONDR < 0.4). A standardized solubility score > 0.45 (range 0-1) is considered suitable for soluble expression.

[0063] The immune stimulation simulation process in this invention is as follows: Three-dimensional agent-based immune stimulation simulation is performed using C-ImmSim v10.3.0 (website: github.com / C-ImmSim), and the vaccine is evaluated using a physiological-scale lymph node compartment model. A three-dose vaccination regimen (0 / 4 / 8 weeks) is adopted, with FASTA format sequences input and a time resolution set to 8 hours / simulation unit. Simulation parameters include 1000 host equivalent units, 200 steps / injection cycle, and tracking of germinal center response (85% confidence interval).

[0064] The secondary and tertiary structure prediction and modeling process in this invention is as follows: The secondary structure of the protein is quantified using the SOPMA (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa_sopma.html) self-optimization prediction method (window=17; similarity threshold=8). The tertiary structure is generated using the AlphaFold2v2.3.0 multimer model, optimized by the AMBER force field, and after 5 cycles, all models are subjected to 10 ns molecular dynamics equilibrium in explicit solvent.

[0065] The structural optimization and validation process in this invention is as follows: The initial AlphaFold2 model was optimized using the loop modeling protocol (ModLoop algorithm) and SCWRL4 sidechain rearrangement of GalaxyWEB2024Q2 (website: galaxyweb.com). Structural quality validation metrics included: ERRAT overall quality score (>85% acceptable region), Verify3D compatibility (>80% residue score >0.2), PROCHECK Laplace plot analysis (<2% outliers), and MolProbity conflict score (<10 conflicts / 100 residues). The final model achieved a QMEANDisCo global score >0.7, with local Z-values ​​within ±2.0 in each domain.

[0066] The molecular docking and dynamics analysis process in this invention is as follows: Molecular docking studies were conducted using the HDOCK v2.0 server (http: / / hdock.phys.hust.edu.cn / ) to evaluate the interaction between the engineered h-tFc-MEV vaccine and human FcRn (PDB number: 1EXU). Indicators included the interface ΔG score calculated using the PISA algorithm, hydrogen bond network, and solvent-accessible surface analysis visualized in PyMOL v2.5.0. Gromacs 2022.3 software was used for molecular dynamics simulations. For small molecule pretreatment, a GAFF force field was applied to the small molecules using AmberTools, and hydrogenation and RESP potential energy calculations were performed using Gaussian 16W. The potential energy data were integrated into the topology file of the molecular dynamics system. Simulations were conducted at a static temperature of 300 K and atmospheric pressure (1 Bar). An Amber99sb-ildn force field was applied, with water molecules as the solvent (Tip3p water model), and the total charge of the simulation system was neutralized by adding an appropriate amount of Na+ ions. Energy minimization was performed using the steepest descent method, followed by isothermal-isochoric (NVT) and isothermal-isobaric (NPT) ensemble equilibrations, each for 100,000 steps, with a coupling constant of 0.1 picoseconds and a duration of 100 picoseconds. Finally, free molecular dynamics simulations were performed, totaling 5,000,000 steps at a step size of 2 femtoseconds, for a total of 100 nanoseconds. After the simulations, the trajectory was evaluated using the software's built-in analysis tools, calculating the root mean square deviation (RMSD), root mean square fluctuation (RMSF), and protein rotation radius for each amino acid trajectory. Simultaneously, free energy calculations (MMGBSA) and free energy topological analysis were performed.

[0067] The recombinant plasmid construction process in this invention is as follows: Youkang Company constructs MEV and h-tFc-MEV recombinant plasmids through restriction cloning: using pET-28a plasmid as a template, the MEV and h-tFc-MEV genes are amplified by PCR, and the fragment containing the target gene is integrated into the pET-19b plasmid vector through NdeI and XhoI restriction sites.

[0068] The protein expression and purification process in this invention is as follows: The target gene was cloned into the pET-19b vector and then transformed into competent *E. coli* BL21(DE3) cells. Expression was induced at 18°C ​​for 20 hours using 0.5 mM IPTG (Sigma-Aldrich, I6758; Merck KGaA, Darmstadt, Germany). The bacterial pellet was resuspended in lysis buffer (containing 20 mM Tris-HCl pH 8.0 (Sigma-Aldrich T6066; Merck KGaA, St. Louis, Missouri, USA), 300 mM NaCl (Absin, 47052148; Abogen Biosciences, Shanghai, China), 10 mM imidazole (Absin I5513; Abogen Biosciences, Shanghai, China) and a mixture of protease inhibitors (Solarbio P8340; Solarbio, Beijing, China)), and after sonication, centrifuged (12,000 × g, 30 min). The supernatant was then loaded onto a nickel-NTA affinity chromatography column (Cytiva 17524802; Cytiva Corporation, Marburg, Massachusetts, USA), washed with buffer containing 50 mM imidazole, and eluted under native conditions using a linear gradient of imidazole (50 mM to 300 mM). The target protein was eluted at a concentration of 150 mM imidazole. After desalting, the sample was stored at -80°C.

[0069] The preparation and characterization method of the chitosan nanovaccine in this invention is as follows: MEV and h-tFc-MEV nanoparticles were prepared by chitosan ionic crosslinking. Specific steps: Chitosan (1 mg / mL, Hengxing, Zhenjiang, Jiangsu, China) was dissolved in 1% acetic acid solution (LINSHIHUAXUE 64-19-7, Linyi, Shandong, China) and filtered through a 0.45 μm filter membrane. Under magnetic stirring, 5 mL of chitosan solution (pH 4.6) was mixed with 1.25 mL of a recombinant protein solution of equal concentration (1 mg / mL), followed by slow addition of 1.25 mL of sodium tripolyphosphate (1 mg / mL, Hengxing, Zhenjiang, Jiangsu, China). The mixture was stirred continuously at room temperature for 1 hour to complete gelation. The mixture was centrifuged (10,000 g, 30 min) to separate the nanoparticles, washed, and recovered to ensure the purity and integrity of the nanoparticles.

[0070] The animal immunization process in this invention is as follows: Female BALB / c mice aged 6-8 weeks and weighing 18-22 grams were randomly divided into three groups and administered 200 μL of chitosan nanoparticles carrying h-tFc-MEV (70 μg / dose), MEV protein (70 μg / dose), or a PBS control on days 0, 14, and 28, respectively. At the specified endpoints (day 42: 14 days after final immunization; day 180: persistence assessment at 6 months), mice were sacrificed by carbon dioxide asphyxiation, and spleen, mesenteric lymph nodes, lamina propria, and serum were immediately collected. This study was approved by the Animal Care and Use Committee (ACUC) of Xinjiang Medical University, protocol number K202409-17.

[0071] The enzyme-linked immunosorbent assay (ELISA) procedure in this invention is as follows:

[0072] Serum sample collection and preparation: Blood was collected from the posterior orbital plexus of BALB / c mice under isoflurane anesthesia. After coagulation at room temperature for 30 minutes, serum was separated by centrifugation (2,000×g, 15 min, Eppendorf 5804R, Germany). Hemolyzed samples were discarded.

[0073] Mucosal irrigation solution

[0074] Bronchoalveolar lavage fluid (BALF): The lungs were lavaged three times with 1 mL of sterile PBS. The combined fluid was centrifuged at 800 × g for 10 min at 4 °C to remove cell debris. Intestinal lavage fluid: The small intestine was flushed with 5 mL of ice-cold PBS. The effluent was centrifuged at 3,000 × g for 15 min to precipitate mucus. Vaginal / nasal lavage fluid: Collected with 1 mL of PBS, centrifuged at 2,000 × g for 10 min, and filtered through a 0.22 μm membrane (Millipore, Billerica, Massachusetts, USA). All samples were aliquoted and stored at -80 °C. Pre-dilution optimizations were as follows: BALF (1:5–1:20), vaginal lavage fluid (1:2–1:10).

[0075] h-tFc-MEV or recombinant MEV protein (2.5 μg / mL in PBS) was coated onto a high-binding 96-well plate (Costar 3590, Corning) at 50 μL per well and incubated overnight at 4°C. After three washes with PBS containing 0.05% Tween-20 (PBST), the plate was blocked with 5% (w / v) skim milk in PBST at room temperature for 1 hour. Serially diluted serum samples (serially diluted in blocking buffer) were then added and incubated for 2 hours. After washing with PBST, the plates were incubated for 1 hour with horseradish peroxidase (HRP)-labeled antibodies: goat anti-mouse IgA (BioLegend 407002, San Diego, California, USA; 1:5,000), IgG (BioLegend 405306, San Diego, California, USA; 1:1,000), IgG2a (SouthernBiotech 1081-05, Birmingham, Alabama, USA; 1:6,000), or IgG1 (SouthernBiotech 1071-05, Birmingham, Alabama, USA; 1:8,000). After incubation with the substrate for 45 minutes (37°C, protected from light), the sample was developed with 3,3',5,5'-tetramethylbenzidine (BioLegend 421101, San Diego, CA, USA) for 10 minutes, and the reaction was terminated with 2M H2SO4 (Sigma-Aldrich 258105, St. Louis, Missouri, USA). Absorbance was measured at 450 nm (reference wavelength 630 nm) using a SpectraMax M5 microplate reader (Sigma-Aldrich, 258105; St. Louis, Missouri, USA). The endpoint antibody titer was defined as the highest value of the serum dilution with an optical density (OD450) ≥ 0.5, as previously described 36. Serum IL-21 concentrations were determined using a commercially available mouse IL-21 ELISA kit (Sigma-Aldrich 258105, St. Louis, Missouri, USA) according to the manufacturer's protocol.

[0076] The cell loss process in this invention is as follows: Two weeks after final immunization, spleen single-cell suspensions are prepared by gentle mechanical dissociation. Cells are stimulated with 5 μg / mL h-tFc-MEV or MEV recombinant protein for 16 hours and incubated at 37°C and 5% CO2, followed by incubation with GolgiPlug (BD Biosciences 555029, Franklin Lake, NJ, USA) for 4-6 hours to inhibit protein transport. Before staining, Fc receptors are blocked using anti-CD16 / CD32 antibody (BD Biosciences 553142, Franklin Lake, NJ, USA). Surface markers are labeled with fluorescently labeled antibodies, followed by Cytofix / Cytoperm.TM Cells were fixed and permeabilized in a solution (BD Biosciences 554714, Franklin Lake, NJ, USA) for intracellular IFN-γ and IL-4 antibody staining. After washing, cells were resuspended in FACS buffer (PBS containing 1% FBS [Gibco 26140079, Glen Island, NY, USA] and 0.1% sodium azide [Sigma-Aldrich S2002, Darmstadt, Germany]) and subjected to flow cytometry (BD LSR Fortessa). TM (BD Biosciences, Franklin Lake, New Jersey, USA) Analysis was performed. Simultaneously, mesenteric lamina propria cells were isolated by enzymatic digestion, while spleen cells were prepared by mechanical dissociation. Phenotypic analysis of dendritic cell (DC) subsets (CD11c) was performed using specific fluorescently labeled antibodies. + MHC Class II + Six months post-immunization, single-cell suspensions of lymph nodes and spleen were prepared via gentle mechanical dissociation. These suspensions were analyzed using CXCR5. + PD-1 + Follicular helper T cells (TFH) were identified by staining using GL7. + FAS + Biomarkers for identifying germinal center B cells (GCBs) via CD44 + CD62L + Expression was used to identify central memory T cells (TCMs). Cells and antibody mixtures were incubated at 37°C in the dark for 20 minutes. The expression was performed using the BD FACSLyric system (normalized voltage settings and...). Compensation matrices validated by CompBeads (BD Biosciences, Franklin Lake, NJ 552843, USA) were used to acquire flow cytometry data. FlowJo v10.8 software (BD Life Sciences, Ashland, Oregon, USA) was used for data analysis, employing a pre-established gating strategy to ensure continuity of analysis across all experimental time points.

[0077] plan

[0078] The following antibodies were used for IFN-γ detection: CD45-APC-cy7 (BD Biosciences 557659, Franklin Lake, New Jersey, USA), CD3-APC (Biolegend 100236, San Diego, California, USA), CD4-FITC (Biolegend 100406, San Diego, California, USA), CD8-APC-Cy7 (BD Biosciences 557654, Franklin Lake, New Jersey, USA), and IFN-γ-V500 (BD Biosciences 561980, Franklin Lake, New Jersey, USA).

[0079] The following antibodies were used for IL-4 detection: CD45-APC-cy7 (BD Biosciences 557659, Franklin Lake, New Jersey, USA), CD3-APC (Biolegend 100236, San Diego, California, USA), CD4-FITC (Biolegend 100406, San Diego, California, USA), CD8-APC-Cy7 (BD Biosciences 557654, Franklin Lake, New Jersey, USA), and IL-4-V450 (BD Biosciences 560701, Franklin Lake, New Jersey, USA).

[0080] The following antibodies were used to detect dendritic cells (DCs): Live / dead-Bv421 (Thermo Fisher Scientific L34955, Waltham, Massachusetts, USA), CD45-APC-cy7 (BD Biosciences 557659, Franklin Lake, New Jersey, USA), Lin-APC (BioLegend 133306, San Diego, California, USA), CD11c-PE-cy7 (BD Biosciences 558079, Franklin Lake, New Jersey, USA), and MHC-II-FITC (Biolegend 116406, San Diego, California, USA).

[0081] The following antibodies were used to detect central memory T cells (TCM): CD3-APC (Biolegend 100236, San Diego, California, USA), CD4-FITC (Biolegend 100406, San Diego, California, USA), CD8-APC-Cy7 (BD Biosciences 557654, Franklin Lake, New Jersey, USA), CD44-PerCP-Cy5.5 (BD Biosciences 1278947, Franklin Lake, New Jersey, USA), and CD62L-PE-Cy7 (BD Biosciences 1175634, Franklin Lake, New Jersey, USA).

[0082] The following antibodies were used to detect follicular helper T cells (TFH): CD45-APC-cy7 (BD Biosciences 557659, Franklin Lake, NJ, USA), CD3-APC (Biolegend 100236, San Diego, CA, USA), CD4-FITC (Biolegend 100406, San Diego, CA, USA), CXCR5-PE (BD Biosciences 1165377, Franklin Lake, NJ, USA), and PD-1-Bv605 (BD Biosciences 748267, Franklin Lake, NJ, USA).

[0083] The following antibodies were used to detect germinal center B cells (GCB): CD45-APC-cy7 (BD Biosciences 557659, Franklin Lake, New Jersey, USA), CD3-APC (Biolegend 100236, San Diego, California, USA), B220-FITC (Biolegend 103205, San Diego, California, USA), GL7-PE (Biolegend 144607, San Diego, California, USA), and FAS-Bv711 (BD Biosciences 740716, Franklin Lake, New Jersey, USA).

[0084] The immunofluorescence staining procedure in this invention is as follows: Bone marrow-derived dendritic cells were stimulated with 5 μg / mL h-tFc-MEV or recombinant MEV protein for 24 hours, and then seeded onto poly-L-lysine-coated coverslips (Sigma-Aldrich, P4707, St. Louis, Missouri, USA). For fixation, surface antigens were preserved by treatment with 4% p-xyleneformaldehyde (PFA; Sigma-Aldrich P6148, St. Louis, Missouri, USA) at 25°C for 15 minutes, or intracellular antigens were fixed by treatment with pre-cooled methanol (-20°C) for 5 minutes. After washing with PBS, intracellular antigen detection required permeabilization with 0.1% Triton X-100 (Sigma-Aldrich T8787, St. Louis, Missouri, USA) for 10 minutes. The sections were then blocked with 5% bovine serum albumin (BSA; Solarbio A8020, Beijing, China) for 1 hour, followed by incubation at 37°C for 1 hour with cell membrane markers (Thermo Fisher Scientific C10608, Waltham, Massachusetts, USA) and anti-His-tagged primary antibody (STARTER S0B0006, Wuhan, Hubei, China; 1:200 dilution). Subsequently, under light-protected conditions, the sections were incubated with either Alexa Fluor 488- (Invitrogen A-11001, Carlsbad, California, USA; 1:500) of the matched species or Cy3-labeled secondary antibody (Jackson ImmunoResearch, 712-165-153, Westgrove, Pennsylvania, USA; 1:500). Imaging was performed using a Nikon A1R confocal microscope (60× oil immersion lens), capturing Z-stack sections at 0.5 μm intervals and processing with NIS-Elements AR 5.21.

[0085] The protective immune assessment process in this invention is as follows: To assess protective efficacy, all experimental groups were challenged orally on day 42 post-immunization with 10 log10 colony-forming units (CFU) of Brucella strain 16M (ATCC 23456). Animals were euthanized by carbon dioxide asphyxiation on day 56 (14 days post-challenge), and liver, spleen, lung, and mesenteric lymph node tissues were aseptically collected. Tissue homogenates (10% w / v in sterile PBS) were serially diluted 5-fold and cultured on 5% defibrinated sheep blood agar (BD Biosciences 221780, Franklin Lake, NJ, USA) under microaerophilic conditions (5% CO2, 37°C). After 72 hours of incubation, Brucella-specific colonies were counted using an automated colony counter. Data were normalized to organ weight and expressed as log10 CFU / g ± SEM (n = 6 / group).

[0086] Example 1: Design of recombinant antigen h-tFc-MEV

[0087] 1.1 Selection of candidate epitopes for target proteins

[0088] To ensure the safety and efficacy of candidate antigens, homology analysis of Omp25 and VirB10 against human proteins (Homo sapiens, taxid: 9606) was performed using the BLAST program. The results showed that these antigens had no significant homology with human proteins, thus greatly reducing the potential risk of immunogenicity. During antigen design optimization, SignalP software was used to predict signal peptide characteristics. Omp25 showed a Sec / SPI type signal peptide cleavage site between amino acid residues 23 and 24 (AFA-AD motif) with a confidence level of 0.9303, while VirB10 did not show any detectable signal peptide domains. Given the important biological role of signal peptides in guiding nascent proteins to the endoplasmic reticulum, the 1-23 amino acid signal peptide sequence of Omp25 was ultimately removed during recombinant expression to ensure proper localization and functional integrity in host cells. To systematically evaluate the B-cell epitope characteristics of Omp25 and VirB10, this study employed a multi-strategy prediction approach: (1) 16-mer linear epitopes were screened using the ABCpred tool, with a focus on evaluating their antigen-binding potential; (2) Structural bioinformatics algorithms on the IEDB platform were applied to analyze the three-dimensional structural data of Omp25 (PDB number: Q45321) and VirB10 (PDB number: Q8YDZ0) from the UniProt database to predict conformational epitopes (Table 2). This dual-analysis strategy aims to comprehensively resolve the B-cell epitope atlas of candidate antigens, providing precise immunological targets for subsequent vaccine design.

[0089] In vaccine development, ensuring stability, biocompatibility, and safety is paramount. Vaccines must maintain structural stability under varying storage and transportation conditions to prevent degradation and preserve immunogenicity. Optimal hydrophilicity and solubility are crucial for rapid in vivo distribution and amplified immune responses. A core design challenge lies in selecting antigenic peptides that elicit a robust immune response while ensuring these epitopes avoid allergic reactions that could interfere with the immune mechanism. Therefore, ideal vaccine epitopes should possess high stability, hydrophilicity, strong antigenicity, and be non-toxic and non-sensitizing. Specific selection criteria include an antigenicity score greater than 0.4 for protective efficacy, a hydrophilicity index less than 0, a stability score less than 40, and rigorous evaluation to exclude toxicity and sensitization risks. Based on these stringent parameters, Supplementary Material-2 summarizes the screening results, identifying six CTL epitopes, five HTL epitopes, one CBE epitope, and eight LBE epitopes, thus laying a solid foundation for vaccine development.

[0090] The following positions were selected, as shown in Table 1:

[0091] Table 1: Table of Positions

[0092]

[0093]

[0094]

[0095] Targeted design of 1.2h-tFc-MEV

[0096] Through computational screening, we identified six cytotoxic T lymphocyte (CTL) epitopes, five helper T lymphocyte (HTL) epitopes, eight linear B cell epitopes (LBEs), and one conformational B cell epitope (CBE). Screening criteria included antigenicity, hydrophilicity, non-toxicity, and non-sensitizing properties. These epitopes were assembled into a multi-epitope vaccine (MEV) using protease-cleavable linkers. The FcRn receptor, based on the binding of its α and β2 chains, specifically recognizes the Fc region of IgG, particularly the CH2-CH3 domain, thanks to interactions of specific amino acid residues. This design utilizes FcRn-mediated transcellular transport for efficient mucosal delivery while maintaining epitope immunogenicity. The targeting design diagram of h-tFc-MEV is detailed below. Figure 1 .

[0097] The FcRn targeting scaffold is derived from human IgG1 Fc (UniProt ID: P01857), chosen for its high affinity for FcγRI and low binding affinity for FcγRIIB. Key modifications include: eliminating C1q binding through E318A / K320A / K322A mutations to prevent complement activation; monomerization through C226S / C229S substitution to disrupt interchain disulfide bonds and prevent dimer formation; and Efgartigimod-inspired FcRn affinity-enhancing mutations (M252Y / S254T / T256E / H433K / N434F), designed using ABDEG technology to enhance pH-dependent FcRn binding, thereby improving stability under physiological pH gradients and prolonging serum half-life. The optimized h-tFc module is fused with MEV via a flexible GGGS linker to form the h-tFc-MEV complex.

[0098] Example 2: Bioinformatics Validation of Recombinant Antigen h-tFc-MEV

[0099] 2.1 Physicochemical property analysis of recombinant antigen h-tFc-MEV

[0100] The physicochemical properties of h-tFc-MEV were systematically evaluated using the ProtParam tool. Antigenicity prediction was performed using VaxiJen 2.0 validation, and AllerTop v3.0 predicted it to be a non-allergenic vaccine protein (see Table 2).

[0101] Table 2. Physicochemical properties of h-tFc-MEV vaccine

[0102]

[0103] 2.2 Solubility analysis of recombinant antigen h-tFc-MEV

[0104] Based on the machine learning platform Protein-SOL (https: / / protein-sol.manchester.ac.uk), the amino acid sequence of h-tFc-MEV was input, and its good solubility in the E. coli expression system was predicted. (See...) Figure 2

[0105] from Figure 2 As can be seen, the red dashed line represents the optimal classification threshold at 58% solubility, and the gray band represents the population average baseline (PopAvrSol = 0.45). The dataset contains three independent sources: E. coli proteome (n = 679), crystallization tendency test set (n = 200), and UniProt derived sequences (n = 1,294), showing a Pearson correlation coefficient (r) of 0.621. The color gradient corresponds to the QuerySol normalized values, where a score > 0.45 (warm tone) indicates above-average solubility relative to experimental observations; the electrostatic potential and hydrophobicity distribution on the protein structure are visualized using the protein-sol patches algorithm. Blue area: positive charge clusters (> +0.5 kT·e⁻¹), red area: negative charge accumulation region (< -0.5 kT·e⁻¹), yellow area: hydrophobic structural domain (Wimley-White interface scale > 3.0). The color scale is dynamically normalized based on the electrostatic potential range calculated from the input structure.

[0106] 2.3 Immunomodulation of recombinant antigen h-tFc-MEV

[0107] Immunoinformatics simulations showed that h-tFc-MEV vaccination elicited balanced immune activation without overregulating dendritic cells (DCs). The number of DCs remained stable at 180 per cubic millimeter, with 20 per cubic millimeter maintaining the active phenotype. Figure 3 This indicates that DC function was neither overactivated nor suppressed. The T cell response exhibited a gradually amplifying characteristic: the TH cell population gradually increased with each of the three immunizations. Figure 3), activated and resting CD4+ T cells reached peak values ​​of 6,200 / mm³ and 4,100 / mm³, respectively. Figure 3 The B-cell response peaked after the third immunization, with a total count and an activated count of 760 cells / mm³. Figure 3 This aligns with robust humoral immunity. In the Brucella infection model, IgG1 dominates pathogen clearance by targeting surface antigens, while IgM mediates early neutralization. h-tFc-MEV-induced IgG titers peaked at 380,000, confirming a Th1-biased immune response, while IgM levels rose synchronously (peak: 400,000). Figure 3 This demonstrated a coordinated, rapid, and sustained defense capability. In the context of Brucella infection, IFN-γ enhances bactericidal activity by activating macrophages, while IL-2 promotes T cell proliferation to strengthen the immune response. Following three doses of the h-tFc-MEV vaccine, levels of IFN-γ, IL-2, IL-4, and antibodies significantly increased. Figure 3 Immunomodulation showed that the vaccine could activate dendritic cells, drive the expansion of CD4+ T cells and B cells, and induce a Th1-dominant immune response (mainly IgG1) and multicellular secretion, confirming its potential as a candidate for an anti-brucellosis vaccine.

[0108] Specifically, Figure 3 It should be noted that: (A) DCs can present antigenic peptides on MHC class I and II molecules. The curves show the breakdown of the total bacterial count into active, resting, and internalized states and their corresponding conditions. (B) Total number of TH cells; (C) TH cell population in each state; (D) Total number of B cells; (E) B cell population in each state; (F) Subdivision of antibodies according to subtype; (G) Concentrations of cytokines and interleukins. D in the inset represents a danger signal.

[0109] 2.4 Prediction of the secondary and tertiary structures of recombinant antigen h-tFc-MEV

[0110] The secondary structure of h-tFc-MEV was predicted using the SOPMA algorithm, and the results showed that it consisted of 20.53% α-helices, 54.06% random coils, and 25.41% extended chains. Figure 4 -A). Subsequently, AlphaFold2 (AF2) was used for tertiary structure prediction, and the model with the highest confidence (pLDDT = 56, pTM = 0.347) was identified and structural optimization was performed. The optimized model was visualized in 3D using Discover Studio software. Figure 4-B). It is worth noting that pLDDT (confidence score per residue, ranging from 0 to 100) and pTM (predicted TM score) together indicate the reliability of the model. Figure 4 -C further illustrates the distribution pattern of hydrogen bonds. Importantly, the composition of the tertiary structure (α-helix: 20.51%, random coils: 54.08%, extended chains: 25.41%) shows a striking consistency with the secondary structure prediction, varying by less than 0.5%. This demonstrates the consistency between the AF2 model and experimental structural analysis, thus validating the accuracy of the tertiary structure prediction.

[0111] Example 3: Synthesis of MEV and h-tFc-MEV recombinant proteins, and verification of chitosan MEV and h-tFc-MEV recombinant proteins.

[0112] 3.1 Expression and purification of MEV recombinant protein and h-tFc-MEV recombinant protein

[0113] Based on the predicted stability and bimodal immune activation properties of h-tFc-MEV, we evaluated its biological functions through in vitro and in vivo experiments. Recombinant MEV and h-tFc-MEV proteins were expressed in *E. coli* pLysS using the pET-19b plasmid system. After induction with 0.5 mM IPTG, the proteins were purified by Ni-NTA affinity chromatography under native conditions using a linear imidazole gradient (elution at 150 mM). Western blot analysis using an anti-His-tagged antibody confirmed the purity of both proteins, showing distinct bands at 46 kDa (MEV) and 71.54 kDa (h-tFc-MEV) that closely matched the theoretical molecular weights calculated based on the amino acid sequences. Figure 5 These results indicate the structural integrity of the recombinant protein and accurate post-translational processing. Figure 5 As shown, Figure 5 In the image, A shows the first lane: prestained protein marker (kDa); the second lane: Western blot analysis using an anti-His tag antibody shows a 46kDa band, consistent with the predicted molecular weight of the MEV-His fusion protein. Figure 5 In the image, lane B shows the first lane: prestained protein marker (kDa); lane 2: Western blot analysis using an anti-His tag antibody shows a 71.54 kDa band, consistent with the predicted molecular weight of the h-tFc-MEV-His fusion protein.

[0114] 3.2 Chitosan-based nanoparticle system engineering to enhance mucosal vaccine delivery

[0115] Comprehensive characterization using dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed the key advantages of engineered h-tFc-MEV-CS and MEV-CS nanoparticles. Morphologically, both nanoparticles exhibited a regular spherical shape. Despite an increase in particle size over time, storage at 4°C significantly improved stability, superior to room temperature (25°C). Figure 6 -Aa,d). Especially in the early stages after preparation (0-8 hours), the average particle size of chitosan nanoparticles can be controlled within 200 nanometers. Figure 7 The nanoparticles, with their -Ba,b,d,e) structure, meet the basic size requirement (<200 nm) for effective penetration of intestinal mucus, providing a solid physical foundation for subsequent delivery. Regarding the polydispersity index (PdI), the trend of PdI changes over time indicates that the PdI fluctuation of nanoparticles stored at 4°C is significantly smaller than that at room temperature (25°C), highlighting their significant stability advantage. In the early stages after preparation (0-8 hours), the PdI of chitosan nanoparticles remained stably below 0.3, a key piece of evidence for their ideal nanoparticle vaccine properties, indicating that a relatively uniform particle distribution can be maintained during delivery, thereby promoting the smooth activation of subsequent immune responses. In terms of charge properties, the surface charge intensity of the nanoparticles remained relatively stable during the seven-day observation period, with only a slight decrease, and the overall surface charge was positive. This again demonstrates that the stability at 4°C is superior to that at room temperature (25°C). Particularly in the early stages after preparation (0-8 hours), the zeta potential of the chitosan nanoparticles reached +33.3±1.45 mV and +32.2±1.15 mV (…). Figure 7 -Bc,f). This positive charge enables the nanoparticles to easily attach to the mucosa through electrostatic interactions with the negatively charged epithelial surface, significantly promoting cell internalization and creating favorable conditions for effective antigen presentation, potentially triggering a more robust immune response. Furthermore, the protein loading efficiencies of h-tFc-MEV and MEV are also satisfactory, reaching 68.52±2.31% and 67.36±1.97%, respectively. This ensures that a sufficient amount of antigen can be safely and stably loaded onto the nanoparticles to meet the needs of subsequent immune activation. In summary, the newly prepared chitosan nanoparticles, with their nanoscale size, excellent surface charge properties, and superior loading capacity, have the potential to serve as an effective mucosal vaccine delivery system.

[0116] Example 4: Enhanced mucosal penetration ability of chitosan h-tFc-MEV in an oral delivery model

[0117] To evaluate the mucosal penetration ability of h-tFc-MEV protein, we established an oral delivery model using chitosan-based nanocarriers. Mice were randomly divided into three groups and administered 70 μg of chitosan nanoparticle-encapsulated h-tFc-MEV protein, MEV protein, or PBS via oral gavage, respectively. Serum antigen levels were measured using a high-sensitivity ELISA at 8 hours post-administration. Figure 8 The results showed that, quantitative analysis revealed that, compared with the MEV group, the serum protein concentration in the h-tFc-MEV group was significantly increased (3.63-fold increase, p<0.01); Figure 8 This result indicates that h-tFc-MEV has superior transmucosal transport efficiency.

[0118] Example 5: Enhancing Humoral Immunity

[0119] Serological analysis 14 days post-immunization showed that the total antigen-specific IgG titer in the chitosan h-tFc-MEV group was 1.54 times higher than that in the chitosan MEV group (p<0.01). Figure 9 This finding, combined with the significant increase in total antibody levels, indicates that h-tFc-MEV comprehensively enhances the strength of the antibody response.

[0120] Example 6: FcRn-mediated subunit vaccine strategy significantly improves antigen presentation efficiency

[0121] FcRn targeted delivery strategies have become a key approach in subunit vaccine design. By enhancing the recycling of IgG-mediated immune complexes within dendritic cells (DCs), they amplify MHC I and MHC II antigen presentation pathways, activating CD8+ and CD4+ T cell responses.51 In vitro experiments showed that DCs treated with chitosan h-tFc-MEV exhibited 2.02 times higher antigen presentation efficiency after 24 hours of incubation compared to the chitosan MEV-treated group. Figure 10 The phenomenon (-AB) is mechanistically related to FcRn-mediated optimized endocytosis and transport. Subsequent in vivo validation using flow cytometry analysis showed that chitosan h-tFc-MEV immunotherapy induced a 2.28-fold, 3.11-fold, and 2.60-fold increase in the mature dendritic cell (DC) populations in the spleen, small intestine, and large intestinal lamina propria, respectively. Figure 10 -CD; sorting strategy in supplementary material-12), while the production of IL-12 in spleen tissue increased by 1.41 times ( Figure 10 These consistent results confirm that FcRn-driven vaccine engineering not only enhances the antigen-presenting capacity of dendritic cells (DCs) but also systematically activates key effector cells.

[0122] Specifically, Figure 10(A) This figure shows immunofluorescence images of dendritic cells (DCs) treated with h-tFc-MEV or MEV. (B) Semi-quantitative analysis of fluorescence intensity ratios was performed using ImageJ (normalizing the fluorescence intensity of h-tFc-MEV or MEV to the total fluorescence intensity) (n=3 per group). (C) Representative flow cytometry plots show the populations of mature dendritic cells (CD11c+MHC II+) in the lamina propria of the spleen and intestinal mucosa; sorting strategies are detailed in Supplementary Material-7. (D) Quantitative analysis of the frequency of mature dendritic cells in the lamina propria of the spleen and intestinal mucosa is shown. (E) IL-12 levels in spleen homogenate were measured by ELISA. Data are presented as mean ± standard error (n=6 / group), and the height of the bars represents the group mean. Differences between groups were assessed using one-way ANOVA and Tukey multiple comparisons after validating the normality of the data using the Shapiro-Wilk test. Statistical analysis was performed using GraphPad Prism software.

[0123] Example 7: Chitosan h-tFc-MEV stimulates pathogen-specific T-cell immunity

[0124] h-tFc-MEV vaccine significantly enhances T-cell-mediated immune protection by mimicking pathogen infection. Flow cytometry analysis showed that 14 days after final immunization, h-tFc-MEV immunization resulted in a pronounced Th1-type immune profile in spleen cells: compared to the MEV group, IFN-γ... + CD4 + and CD8 + T cell frequencies increased by 2.52-fold and 2.44-fold, respectively. Figure 11 Notably, Th2 polarization was also observed simultaneously with IL-4. + CD4 + and CD8 + T cells increased by 2.65-fold and 5.66-fold, respectively. Figure 11 Notably, h-tFc-MEV overcomes the limitations of MEV (the MEV group and the PBS group showed differences in CD8). + IL-4 + (No significant differences were found in T cell responses), demonstrating its dual regulatory capacity on Th1 / Th2 immunity. These findings highlight the role of FcRn targeted delivery in optimizing CD4+. + / CD8 + Its advantages in T cell balance and Th1 / Th2 synergy can effectively resist Brucella.

[0125] Example 8: FcRn targeting strategy enhances long-term immune memory induced by h-tFc-MEV vaccine: the key role of central memory T cells and germinal center activity (where h-tFc-MEV and MEV are both encapsulated in chitosan).

[0126] FcRn targets mucosal immunity and significantly enhances the level of antigen-specific central memory T cells (TCMs), maintaining a high level for at least six months after booster immunization, while also maintaining a high frequency of germinal center B cells (GCBs) and follicular helper T cells (TFHs)—key cellular factors for strong and durable immune memory.

[0127] TCM cells play a crucial role in vaccine-induced long-term memory. Upon re-exposure to the antigen, these cells are rapidly activated to amplify the immune response, thereby ensuring a robust defense. In mice vaccinated with the h-tFc-MEV vaccine, CD4+ cells... + (7.79±0.91%) and CD8 + (40.90±6.22%) Persistent amplification of the TCM subset persisted 180 days post-immunization, exceeding the levels in the PBS control group by 2.31-fold and 3.07-fold, respectively. Compared to the MEV-immunized control group, these values ​​increased by 2.06-fold and 1.51-fold, respectively. Figure 12 The continued presence of TCM suggests that the recall response is accelerated during potential Brucella exposure, thereby enhancing protective efficacy.

[0128] In an in-depth evaluation of vaccine efficacy, the immune dynamics in lymphoid organs of mice vaccinated with h-tFc-MEV were systematically monitored at two and six months. The study showed sustained activation of germinal centers in lymph nodes and spleen, where TFH and GCB cells established a stable immune synergy through an IL-21-mediated paracrine network. This cell-cell interaction promoted B cell clonal expansion, plasma cell differentiation, and the formation of a memory B cell pool, thus supporting long-term immunity. Flow cytometry analysis performed six months after immunization showed that the proportion of TFH cells in lymph nodes in the h-tFc-MEV group was (18.00±3.17)%. Figure 13 ),

[0129] In the spleen, it was (12.36±2.00)%. Figure 13 Compared with the PBS control group, the levels of GCB cells increased by 7.40-fold and 6.31-fold, respectively, and compared with the MEV control group, they showed advantages of 1.81-fold and 1.95-fold, respectively. Meanwhile, GCB cells reached (0.98±0.19)% in lymph nodes. Figure 14 In the spleen, the percentage was (2.70±0.31)%. Figure 14The levels of IL-21 in the spleen were increased by 4.98-fold and 28.8-fold compared to the PBS group, and improved by 2.03-fold and 3.05-fold compared to the MEV group. ELISA confirmed elevated IL-21 levels in the spleen (545.8±64.3 pg / mL). Figure 15 This is associated with the expansion of TFH and GCB cells. Serum analysis ( Figure 15 The results showed that the h-tFc-MEV group maintained a high level of protective IgG, highlighting its dual advantages in promoting early rapid response and establishing lasting immune memory.

[0130] Example 9: Mucosal immune response induced by h-tFc-MEV vaccine via FcRn targeting strategy (where h-tFc-MEV and MEV are both encapsulated by chitosan).

[0131] The production of mucosal-specific antibodies in mucosal secretions is a key component of the mucosal immune response. Two weeks after vaccination, the levels of vaccine-specific IgG and IgA in intestinal lumen, bronchoalveolar, nasal, and vaginal lavage fluids were precisely quantified by ELISA. Results showed that, compared with the PBS group, the h-tFc-MEV vaccine significantly enhanced the production of IgA and IgG in the intestinal and lung mucosa (P < 0.01), indicating a strong proximal mucosal immune response. Notably, compared with MEV, h-tFc-MEV induced significantly higher levels of IgA and IgG in the lung mucosa (P < 0.01), while the increase in IgA / IgG in the intestinal mucosa was statistically weaker (P > 0.05). In nasal lavage fluid, compared with PBS, h-tFc-MEV induced an increase in IgG and IgA (P < 0.01), but there was no significant difference compared with MEV (P > 0.01). IgG titers were detectable in vaginal secretions, with the h-tFc-MEV group showing a moderately higher titer than PBS (P < 0.05), but no difference compared to MEV. Furthermore, compared to PBS, h-tFc-MEV significantly enhanced IgA in the vaginal mucosa (P < 0.01), but this difference was not significant compared to MEV (P > 0.01). These findings suggest that h-tFc-MEV tends to activate local mucosal IgA responses in the gastrointestinal and bronchial-associated lymphoid tissues, while providing limited protection in distal mucosal sites, highlighting the regional specificity of mucosal immunity. Figure 16 )

[0132] Example 10: Chitosan h-tFc-MEV vaccine challenge experiment (where h-tFc-MEV and MEV are both encapsulated by chitosan).

[0133] Mice immunized with three doses were orally challenged with 10.3 log10 CFU of Brucella. Bacterial load in tissues was quantified by plate culture 14 days post-challenge. Compared to the PBS control group, the mesenteric lymph node bacterial load was reduced by 1.7 log10 CFU / g in the h-tFc-MEV group, and by 0.77 ± 0.43 log10 CFU / g compared to the MEV group (p < 0.01), indicating that the vaccine specifically inhibits the spread of enteropathogens.

[0134] In terms of systemic protection, the h-tFc-MEV vaccine showed significant efficacy: the bacterial load in the spleen (2.365±0.62log10 CFU / g) was reduced by 28.2%±19.1% compared to the MEV control group (p<0.01), and the bacterial load in the liver (1.62±0.34log10 CFU / g) was reduced by 25.7%±17.4% (p<0.01). Lung protection was tissue-specific, reducing the bacterial load by 39.5%±26.4% compared to the PBS group (PBS group: 2.76±0.25log10 CFU / g; p<0.01), but only by 5.7%±64.3% compared to the MEV group (MEV group: 1.77±0.91log10 CFU / g; p>0.01). The renal protective effect was stratified. Compared with the PBS group, bacterial clearance reached 72.7% ± 24.5% (PBS group: 2.49 ± 0.37 log10 CFU / g; p < 0.01), and decreased by 46.7% ± 75.3% compared with the MEV group (MEV group: 1.28 ± 0.63 log10 CFU / g; p > 0.01). Figure 17 These data highlight the comprehensive protective efficacy of FcRn-targeted mucosal vaccines.

[0135] The h-tFc-MEV vaccine can elicit a dual-system mucosal immune response, effectively blocking Brucella invasion via the gastrointestinal and respiratory tracts, and establishing a strong protective effect in organs of the reticuloendothelial system (spleen, liver, and lungs), exerting its effect by inhibiting pathogen colonization and replication.

Claims

1. A Brucella multi-epitope peptide, characterized in that: The multi-epitope peptide is composed of the following peptide sequences linked together: amino acid sequences 56-65 of OMP25, amino acid sequences 57-66 of OMP25, amino acid sequences 163-171 of OMP25, amino acid sequences 71-80 of VirB10, amino acid sequences 72-81 of VirB10, amino acid sequences 79-88 of VirB10, amino acid sequences 49-63 of OMP25, amino acid sequences 50-64 of OMP25, amino acid sequences 306-320 of VirB10, and amino acid sequences 265-279 of VirB10. The sequences of the following amino acids are listed: amino acid sequence of VirB10 (positions 366-380), amino acid sequence of OMP25 (positions 45-60), amino acid sequence of OMP25 (positions 165-180), amino acid sequence of OMP25 (positions 182-197), amino acid sequence of VirB10 (positions 235-250), amino acid sequence of VirB10 (positions 175-190), amino acid sequence of VirB10 (positions 309-324), amino acid sequence of VirB10 (positions 83-98), amino acid sequence of VirB10 (positions 280-295), and amino acid sequence of VirB10 (positions 122-239).

2. A Brucella multiepitope vaccine, characterized in that: The multi-epitope vaccine comprises the multi-epitope peptide and h-tFc protein as described in claim 1, wherein the h-tFc protein is added to the N-terminus of the multi-epitope peptide as described in claim 1, and the h-tFc and the multi-epitope peptide are linked by GGGS to form a recombinant protein.

3. The multi-epitope vaccine as described in claim 2, characterized in that: The h-tFc is obtained by modifying FcRn, including the following modifications: E318A, K320A, K322A, C226S, C229S, M252Y, S254T, T256E, H433K, N434F; the amino acid sequence of h-tFc is shown in SEQ ID NO.

1.

4. The multi-epitope vaccine as described in claim 3, characterized in that: The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

2.

5. A recombinant nucleic acid molecule, characterized in that: The recombinant protein of claim 4 is encoded, wherein the sequence of the recombinant nucleic acid molecule is shown in SEQ ID NO.

3.

6. A chitosan nanovaccine, characterized in that: The multi-epitope vaccine as described in any one of claims 2-4 is encapsulated by chitosan.

7. A method for preparing a chitosan nanovaccine as described in claim 6, characterized in that: Includes the following steps: (1) Dissolve chitosan in acetic acid and filter; (2) Mix the chitosan solution obtained in step (1) with the multi-epitope vaccine as described in any one of claims 2-4, add sodium tripolyphosphate, and stir to complete gelation.

8. The use of the multi-epitope peptide of claim 1, or the multi-epitope peptide vaccine of any one of claims 2-4, or the recombinant nucleic acid molecule of claim 5, or the chitosan nanovaccine of claim 6, in the preparation of a medicament for the prevention or improvement of diseases caused by Brucella.

9. The application as described in claim 8, characterized in that: The drug is administered orally.

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