Brucella recombinant polyepitope vaccine and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a Brucella recombinant multi-epitope vaccine and application thereof. The application provides a Brucella recombinant multi-epitope vaccine protein, and the vaccine protein is a first vaccine variant rBMEV1 or a second vaccine variant rBMEV2. The results of examples prove that the vaccine provided by the application can stimulate a strong cross immune response against main pathogenic species (such as sheep species, bovine species and porcine species) of Brucella. Animal tests prove that the vaccine can induce high-level and long-lasting specific antibodies, and significantly activate CD4 + and CD8 + T cells and corresponding cytokine responses, and has good safety. The application solves the problems of a narrow protection spectrum, a dependence on exogenous adjuvants and a single immune type of a traditional Brucella vaccine, and provides a new generation of high-efficiency, broad-spectrum and safe candidate vaccine strategy for the prevention and control of brucellosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a Brucella recombinant multiepitope vaccine and its application. Background Technology

[0002] Brucellosis is caused by the Gram-negative bacterium Brucella spp. (Brucella) Brucella spp Brucellosis is a zoonotic bacterial disease caused by Brucella. Currently, it has become a global public health concern, posing a threat to the health and safety of both humans and animals. In animals, Brucella infection can lead to epididymitis in males and abortion in pregnant animals, causing significant economic losses to the livestock industry; in humans, it manifests as joint swelling and undulant fever. In China, brucellosis has resurfaced over the past two decades. Monitoring data shows that reported cases in China rose from 38,151 in 2011 to over 70,000 in 2023, with the incidence rate increasing from 2.8 cases per 100,000 people to 5.2 cases per 100,000 people. These trends highlight the growing public health and economic burden of brucellosis.

[0003] Vaccination remains the most important means of controlling animal diseases, and brucellosis vaccines can effectively reduce morbidity. Government data shows that brucellosis vaccination can save the sheep farming industry over 4 billion RMB in economic losses and significantly reduce infection rates in humans and animals. Currently used vaccines include inactivated vaccines, live attenuated vaccines, and subunit vaccines. Among these, live attenuated vaccines (such as Brucella bovis S19 and RB51 strains) remain the most effective and widely used vaccines for controlling brucellosis, as they can more efficiently stimulate the body's cellular immune response and provide long-lasting protection. These vaccines also have advantages such as simple production processes and low cost, but they have drawbacks such as interfering with diagnostic testing, potential pathogenicity in humans, and the possibility of causing abortion in pregnant animals. Although inactivated vaccines have a high safety profile, their immunostimulatory capacity is relatively weak, and routine serological tests cannot distinguish between naturally infected and vaccinated animals, posing challenges to diagnosis.

[0004] There is an urgent need for a novel Brucella vaccine that is highly safe, has strong immunogenicity, and does not interfere with diagnosis. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant multivalent epitope vaccine for Brucella. The vaccine provided by this invention can synergistically activate humoral and cellular immune responses, effectively eliminating intracellular parasitic Brucella.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention discloses a recombinant multi-epitope vaccine protein of Brucella, wherein the vaccine protein comprises a core immunogenic sequence consisting of a series of conserved epitopes of multiple Brucella protective antigens, wherein the core immunogenic sequence consists of 8 cytotoxic T lymphocyte epitopes, 7 helper T lymphocyte epitopes and 8 B cell epitopes connected in series, and a universal helper epitope located at the N-terminus of the core immunogenic sequence; the universal helper epitope has TLR agonist activity.

[0007] Preferably, the amino acid sequences of the eight cytotoxic T lymphocyte epitopes are shown in SEQ ID NO.1-SEQ ID NO.8.

[0008] Preferably, the amino acid sequences of the seven helper T lymphocyte epitopes are shown in SEQ ID NO.9-SEQ ID NO.15, respectively.

[0009] Preferably, the amino acid sequences of the eight B-cell epitopes are shown in SEQ ID NO.16-SEQ ID NO.23, respectively.

[0010] Preferably, the vaccine protein is a first vaccine variant rBMEV1 or a second vaccine variant rBMEV2; wherein the first vaccine variant rBMEV1 has a membrane-penetrating peptide fused to the C-terminus of the core immunogen sequence, and the second vaccine variant rBMEV2 has an immunoglobulin Fc fragment fused to the C-terminus of the core immunogen sequence.

[0011] Preferably, the membrane-penetrating peptide is derived from HIV-1 TAT protein; the immunoglobulin Fc fragment is derived from human IgG Fc fragment.

[0012] Preferably, the amino acid sequence of the first vaccine variant rBMEV1 is shown in SEQ ID NO.24; and the amino acid sequence of the second vaccine variant rBMEV2 is shown in SEQ ID NO.25.

[0013] Preferably, the universal auxiliary epitope with TLR agonist activity is a PADRE sequence, the amino acid sequence of which is shown in SEQ ID NO.26.

[0014] The present invention also provides a pharmaceutical composition comprising the vaccine protein as described above and a pharmaceutically acceptable carrier or adjuvant.

[0015] The present invention also provides the use of the vaccine protein as described above or the pharmaceutical composition as described above in the preparation of a medicament for the prevention of brucellosis.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Providing true broad-spectrum immune protection: The vaccine provided by this invention uses antigenic epitopes derived from 24 classic and prevalent Brucella strains, with highly conserved dominant epitopes selected through immunoinformatics screening. Therefore, the constructed vaccine can avoid triggering cross-immune reactions against multiple Brucella species and strains (including major pathogenic species such as Brucella melitensis, Brucella abortus, and Brucella suis), overcoming the limitation of narrow protection spectrum of traditional single-strain vaccines and providing more reliable broad-spectrum protection against Brucella diversity.

[0017] (2) Achieving a highly efficient and balanced immune response: Through the combination of multiple epitopes, the vaccine provided by this invention can synergistically activate humoral and cellular immune responses: In terms of humoral immunity, vaccination can induce mice to produce high-titer, persistent specific IgG antibodies, and both IgG1 and IgG2a subtypes are significantly increased, indicating that it can simultaneously mobilize Th1 and Th2 type auxiliary immune responses to achieve comprehensive and coordinated humoral immune defense.

[0018] In terms of cellular immunity, the vaccine can effectively promote the specific proliferation of splenic lymphocytes and significantly increase CD4 levels in the spleen. + T cells and CD8 + The proportion of T cells demonstrates that they can induce a strong Th1-type cellular immune response and cytotoxic T lymphocyte (CTL) reaction, which is of great significance for effectively eliminating intracellular parasitic Brucella.

[0019] (3) Excellent self-adjuvant effect, simplified process and enhanced efficacy: This invention introduces a PADRE sequence with TLR activating potential as an internal adjuvant at the N-terminus of the vaccine. Molecular simulations confirm that the vaccine protein can stably bind to TLR2 / TLR4, thereby activating the body's immune system by targeting key innate immune receptors. This design gives the vaccine self-adjuvant properties, enabling it to efficiently activate the innate immune system without relying on or requiring only a small amount of exogenous chemical adjuvants, significantly enhancing the strength and quality of subsequent specific immune responses. This design improves vaccine efficacy while reducing or even avoiding dependence on exogenous chemical adjuvants, which not only helps reduce potential side effects but also simplifies vaccine formulation and manufacturing processes.

[0020] (4) This invention provides two structurally different vaccines (rBMEV1 and rBMEV2), the core difference of which lies in the fusion of a TAT membrane-penetrating peptide and an Fc fragment at the C-terminus, respectively. This differentiated design enables the two vaccines to direct the immune response to a slightly emphased phenotype (such as enhancing intracellular delivery or prolonging the half-life to strengthen the antibody response), thereby providing two complementary candidate strategies for the prevention of brucellosis that can be used alone or in combination, increasing the flexibility to respond to different infection scenarios or host needs.

[0021] (5) Excellent safety profile: The vaccine provided by this invention is a purified recombinant protein, which completely avoids the risks of virulence reversion, danger to pregnant women, and interference with serological diagnosis that are inherent to live attenuated vaccines. Animal experiments show that mice gain normal weight after immunization and there is no pathological damage to major organs, demonstrating its extremely high safety profile and providing a solid foundation for its use in preventive vaccination of healthy populations.

[0022] (6) Solving the diagnostic interference problem and facilitating disease eradication: Since the vaccine provided by this invention is a genetically engineered subunit vaccine, the epitope combination it contains has a designable difference from the complete antigen spectrum of the naturally infected strain. Therefore, a matching differential diagnostic method can be developed based on this to effectively distinguish between vaccinated animals and naturally infected animals, thereby supporting the comprehensive prevention and control strategy of "vaccination-monitoring-eradication", which has significant practical value for eradicating brucellosis in animal husbandry.

[0023] (7) Highly efficient expression and stable preparation process: This invention optimizes the codons of the vaccine gene and uses a mature prokaryotic expression system (pET-28a(+) vector) for production. The resulting recombinant protein has a high expression level and can be expressed using conventional Nitrogen protein expression methods. 2+ Affinity chromatography is a one-step method for efficient purification, with stable processes and controllable costs, and has the potential for large-scale production.

[0024] (8) This invention achieves a comprehensive and organic unity of broad spectrum, high efficiency (self-adjuvant and dual immunization), safety, diagnostic compatibility and production feasibility, and provides a complete and advanced brucellosis vaccine solution from design concept to product preparation. It overcomes the technical defects of existing vaccines and has significant progress and broad application prospects. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1This is a schematic diagram of the predicted protein structures of rBMEV1 and rBMEV2.

[0027] Figure 2 The figures show the results of molecular docking of rBMEV with TLR2 and TLR4, respectively. In the analysis of vaccine molecule-receptor interactions, (AD) show that rBMEV1 interacted with TLR2 (A), rBMEV1 with TLR4 (B), rBMEV2 with TLR2 (C), and rBMEV2 with TLR4 (D), forming 30, 30, 28, and 30 complexes, respectively. The minimum interaction energies obtained from these dockings were -959.8 kcal / mol, -883.2 kcal / mol, -854.1 kcal / mol, and -986.1 kcal / mol, respectively. kcal / mol; (E) At the docking interface, the A chain of TLR2 has 25 hydrogen bonds with the rBMEV1 vaccine structure, and the B chain of TLR2 has 3 hydrogen bonds with the rBMEV1 vaccine structure; (F) The A chain of TLR4 has 28 hydrogen bonds with the rBMEV1 vaccine structure; (G) The A chain of TLR2 has 36 hydrogen bonds with the rBMEV2 vaccine structure, and the B chain of TLR2 has 15 hydrogen bonds with the rBMEV2 vaccine structure; (H) The B chain of TLR4 has 29 hydrogen bonds with the rBMEV2 vaccine structure, and the D chain of TLR4 has 9 hydrogen bonds with the rBMEV2 vaccine structure.

[0028] Figure 3 The following is a diagram showing the results of rBMEV1 immune simulation; (A) the number of B cells of each subtype after vaccination; (B) the production of immunoglobulins after vaccination, and the response of specific subtypes (colored peaks) to the injected antigen (black peaks); (C) the helper T lymphocyte (HTL) population under different conditions; (D) the number of memory HTLs and non-memory HTLs after vaccination; (E) the cytotoxic T lymphocyte (CTL) population under different conditions; and (F) the concentrations of cytokines and interleukins against the vaccine antigen.

[0029] Figure 4 The image shows the results of the rBMEV2 immune simulation, where (A) the number of B cell populations of each subtype after vaccination; (B) the production of immunoglobulins after vaccination, and the response of specific subtypes (colored peaks) to the injected antigen (black peaks); (C) the helper T lymphocyte (HTL) population in different states; (D) the number of memory HTLs and non-memory HTLs after vaccination; (E) the cytotoxic T lymphocyte (CTL) population in different states; and (F) the concentrations of cytokines and interleukins against the vaccine antigen.

[0030] Figure 5 The image shows the double enzyme digestion identification of rBMEV1 and rBMEV2 plasmids.

[0031] Figure 6 The diagram shows the protein purification of rBMEV1 and rBMEV2.

[0032] Figure 7 The diagram shows the potent humoral immune response induced by rBMEV1 and rBMEV2 in mice. (A) Schematic diagram of rBMEV1 / 2 vaccination and sampling timeline; (BD) Serum IgG (B), IgG1 (C), and IgG2a (D) levels in mice immunized with PBS, S2, or rBMEV1 at 14, 28, and 42 DPI; (E) IgG2a / IgG1 ratio in the PBS, S2, and rBMEV1 groups at 42 DPI; (FH) Serum IgG (F), IgG1 (G), and IgG2a (H) levels in mice immunized with PBS, S2, or rBMEV2 at 14, 28, and 42 DPI; (I) IgG2a / IgG1 ratio in the PBS, S2, and rBMEV2 groups at 42 DPI.

[0033] Figure 8 Graphs showing the different Th1 / Th2 polarized cellular immune responses induced by rBMEV1 and rBMEV2 in mice; (A) Schematic diagram of the rBMEV1 / 2 vaccination schedule and subsequent assays to assess cellular immunity; (B) Spleen-to-body weight ratio at 42 DPI; (C) Splenic lymphocyte proliferation stimulation index (SI); (DH) Cytokine levels of IL-2 (D), IFN-γ (E), TNF-α (F), IL-4 (G), and IL-10 (H) in PBS, S2, rBMEV1, and rBMEV2 at 42 DPI; (I, J) CD3+ levels in splenic lymphocytes of the indicator group at 42 DPI. + CD4 + T cells (I) and CD3 + CD8 + Percentage of T cells (J).

[0034] Figure 9 Figure 1 shows the safety evaluation of rBMEV1 and rBMEV2 in a mouse model; (A) Schematic diagram of the rBMEV1 / 2 mouse vaccination protocol; (B) Weight monitoring of immunized mice over time; (C) H&E stained histological sections of heart, liver, spleen, lung, kidney and muscle tissues collected 42 days after immunization. Detailed Implementation

[0035] This invention provides a recombinant multi-epitope vaccine protein of Brucella, the vaccine protein comprising a core immunogenic sequence composed of conserved epitopes of multiple Brucella protective antigens tandemly, the core immunogenic sequence comprising 8 cytotoxic T lymphocyte epitopes, 7 helper T lymphocyte epitopes and 8 B cell epitopes tandemly, and a universal helper epitope located at the N-terminus of the core immunogenic sequence; the universal helper epitope has TLR agonist activity.

[0036] Preferably, the amino acid sequences of the eight cytotoxic T lymphocyte epitopes are shown in SEQ ID NO.1-SEQ ID NO.8.

[0037] Preferably, the amino acid sequences of the seven helper T lymphocyte epitopes are shown in SEQ ID NO.9-SEQ ID NO.15, respectively.

[0038] Preferably, the amino acid sequences of the eight B-cell epitopes are shown in SEQ ID NO.16-SEQ ID NO.23, respectively.

[0039] Preferably, the vaccine protein is a first vaccine variant rBMEV1 or a second vaccine variant rBMEV2; wherein the first vaccine variant rBMEV1 has a membrane-penetrating peptide fused to the C-terminus of the core immunogen sequence, and the second vaccine variant rBMEV2 has an immunoglobulin Fc fragment fused to the C-terminus of the core immunogen sequence.

[0040] Preferably, the membrane-penetrating peptide is derived from HIV-1 TAT protein; the immunoglobulin Fc fragment is derived from human IgG Fc fragment.

[0041] Preferably, the amino acid sequence of the first vaccine variant rBMEV1 is shown in SEQ ID NO.24; and the amino acid sequence of the second vaccine variant rBMEV2 is shown in SEQ ID NO.25.

[0042] Preferably, the universal auxiliary epitope with TLR agonist activity is a PADRE sequence, the amino acid sequence of which is shown in SEQ ID NO.26.

[0043] The present invention also provides a pharmaceutical composition comprising a vaccine protein as described in any of the preceding claims and a pharmaceutically acceptable carrier or adjuvant.

[0044] The present invention also provides the use of the vaccine protein as described in any of the above claims or the pharmaceutical composition described above in the preparation of a medicament for the prevention of brucellosis.

[0045] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0046] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0047] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0048] Example 1: Immuninformatics Design and Molecular Construction of Brucella Broad-Spectrum Multiepitope Vaccine 1.1 Selection of protective antigens and screening of epitopes The complete genome and protein sequences of 24 classic and prevalent Brucella strains (including major pathogenic species such as *Brucella melitensis*, *Brucella abortus*, and *Brucella suis*) were obtained from public databases such as NCBI. Conservation analysis of known Brucella protective antigens (such as OMP25, OMP31, BP26, SOD, and GroEL) was performed using immunoinformatics software (such as IEDB and NetMHC). T-cell epitope prediction: Cytotoxic T-lymphocyte epitopes: The Immunotope Database (IEDB) server was used to predict cytotoxic T-lymphocyte epitopes (CTLs). Using the “IEDB recommended” method, epitopes were predicted based on 9 residues and 72 human leukocyte antigens (HLA). Epitopes were then screened using a TAP score >1.0, IC50 <500 nM, and a proteasome score >1.0. Dominant epitopes derived from a protein with ≥3 HLA alleles and exhibiting high antigenicity (>0.4) in strains carrying the protein were selected. Ultimately, eight highly immunogenic and conserved cytotoxic T-cell (CTL) epitopes were identified. The amino acid sequences of the eight CTLs are as follows: EWQGVPCTF (SEQ ID NO.1), KLTPGYHGF (SEQ ID NO.2), AQGKVRIEY (SEQ ID NO.3), YGVEGDAGY (SEQ ID NO.4), GTVRARLGY (SEQ ID NO.5), TINIPPTLY (SEQ ID NO.6), INIQPIYVY (SEQ ID NO.7), and ASSFNGGIF (SEQ ID NO.8).

[0049] Helper T lymphocyte epitopes: Helper T lymphocyte epitopes (HTLs) were predicted using the online server NetMHCIIpan 4.0. Epitopes were predicted using 27 high-frequency human MHC II (HLA-II) alleles, utilizing a length of 15 amino acid residues. The threshold for strong-binding peptides was set to the default value. Dominant epitopes originated from a protein with ≥3 HLA-II alleles and exhibited high antigenicity (>0.4) in strains carrying this protein. Finally, targeting the MHC II alleles, tools such as NetMHCIIpan were used to predict and screen for 7 high-affinity HTL epitopes. The amino acid sequences are QPIYVYPDDKNNLKE (SEQ ID NO. 9), GVEFRAIREIIMARA (SEQ ID NO. 10), TVKMYEALPTGPGKE (SEQ ID NO. 11), GKVRIEYDGNRLVAD (SEQ ID NO. 12), RVKTPNGVVIDLDSP (SEQ ID NO. 13), VEINMRSIVRGTTSK (SEQ ID NO. 14), and GTGGLAYGKVKSAFN (SEQ ID NO. 15).

[0050] B-cell epitope prediction: Linear B-cell epitopes were predicted using methods such as Kolaskar & Tongaonkar antigenicity assay and BepiPred. Finally, eight dominant epitopes that were highly conserved in most strains and had high prediction scores were screened out. The amino acid sequences of the eight BLEs are as follows: IFETRTT (SEQ ID NO.16), GGKFKHPFSSFDKEDNEQVSGSL (SEQ ID NO.17), VKSAFNL (SEQ ID NO.18), PVQPFLT (SEQ ID NO.19), GHYDPGNTHHHLGPEGD (SEQ ID NO.20), AKTMVQTSTVPMR (SEQ ID NO.21), LKPEEFG (SEQ ID NO.22), and GYSWAKKSKDGLEVK (SEQ ID NO.23).

[0051] 1.2 Design and Assembly of Multiepitope Vaccine Sequences Based on the results of the above screening, the vaccine sequence was designed and assembled: Construction of core multi-epitope tandems: The 8 CTL epitopes, 7 HTL epitopes and 8 B cell epitopes selected above were tandemly linked by connectors ("EAAAK", "KK", "AAY" and "GPGPG").

[0052] Adding functional modules: At the N-terminus of the core sequence, a PADRE sequence with TLR activation potential (13AA SEQ ID NO.26: AKFVAAWTLKAAA) is added.

[0053] Different immune targeting domains were added to the C-terminus of the core sequence to construct two vaccine variants: rBMEV1: Adds the transmembrane peptide domain of the HIV-1 TAT protein to enhance intracellular antigen delivery.

[0054] rBMEV2: Added human IgG Fc to enhance the immunogenicity and half-life of the antigen.

[0055] Each module is separated by a flexible connector, forming complete rBMEV1 and rBMEV2 encoding sequences.

[0056] The sequence of rBMEV1 is EAAAAKAKFVAAWTLKAAAEAAAKEWQGVPCTFAAYKLTPGYHGFAAYAQGKVRIEYAAYYGVEGDAGYAAYGTVRARLGYAAYTINIPPTLYAAYINIQPIYVYAAYASSFNGGIFAAYQPIYVYPDDKNNLKEGPGPGGVEFRAIREIIMARAGPGPGTVKMYEALPTGPGKEGPGPGGKVRIEYDGNRLVADGPGPGRVKTPNGVVIDLDSPGPGPGVEINMRSIVRGTTSKGPGPGGTGGLAYGKVKSAFNGPGPGIFETRTTKKGYSWAKKSKDGLEVKKKGGKFKHPFSSFDKEDNEQVSGSLKKVKSAFNLKKPVQPFLTKKGHYDPGNTHHHLGPEGDKKAKTMVQTSTVPMRKKLKPEEFGKKTGALLAAGAAA (SEQ ID NO. 24); The rBMEV2 sequence is (SEQ ID NO.25).

[0057] 1.3 Bioinformatics Validation After sequence mapping was completed, a series of bioinformatics analyses and simulations were performed on the protein structures of rBMEV1 and rBMEV2 to evaluate their physicochemical properties, self-adjuvant potential, and theoretical immunogenicity.

[0058] 1.3.1 Structural Modeling Tools such as SWISS-MODEL were used to analyze vaccine proteins and predict their tertiary structures.

[0059] Structural predictions show that rBMEV1 consists of 26.44% α-helices, 25.65% extended strands, and 47.91% random coils; while rBMEV2 has a lower proportion of α-helices (14.53%), with extended strands accounting for 27.35% and random coils accounting for 58.12%. These structural features indicate that both antigens are rich in typical "structural antigen" patterns, such as... Figure 1 As shown.

[0060] 1.3.2 Molecular docking and dynamic simulation To verify the function of its built-in adjuvant (PADRE sequence), the predicted models of rBMEV1 and rBMEV2 were molecularly docked with the crystal structures of mouse TLR2 and TLR4, respectively, and molecular dynamics simulations (≥100 ns) were performed using software such as GROMACS.

[0061] The results showed that both vaccine proteins could form stable interactions with the ligand binding pockets of TLR2 / TLR4 with low binding free energy (ΔG), validating their self-adjuvant potential. Figure 2 ).

[0062] 1.3.3 Computer-aided Immunization Simulation The C-ImmSim server was used to predict the immune response after vaccination.

[0063] Simulation results showed that after inoculation with virtual rBMEV1 / rBMEV2, the simulated organism could produce high levels of antibodies (IgM, IgG1, IgG2a) and memory B cells, Th1 / Th2 cells and CTL responses.

[0064] Immune response characteristic analysis showed that ( Figure 3 The vaccine-induced immune response (A, 4A) is primarily mediated by B cells and T cell subsets. During the tertiary immunization process, the body produces significantly elevated antibody titers against the vaccine antigen ( Figure 3 B, 4B), indicating effective activation of the humoral immune response. Further cellular immune analysis showed ( Figure 3 During secondary and tertiary immune responses (C, D, 4C, D), the number of activated helper T cells (TH), total TH cells, and memory TH cells all showed an increasing trend. Simultaneously, cytotoxic T lymphocytes (CTLs) showed a stable increase in number after each immunization. Figure 3 E, 4E). Furthermore, after the completion of the tertiary immunization phase, cytokines such as interferon-γ (IFN-γ) and members of the interleukin family exhibit a potent synergistic response. Figure 3 (F, 4F). Based on the above multidimensional immune index analysis results, it is confirmed that rBMEV1 and rBMEV2 vaccine molecules can effectively activate the adaptive immune system and induce a lasting and comprehensive immune protective effect.

[0065] Example 2: Prokaryotic Expression and Purification of Vaccine Protein 2.1 Gene Synthesis and Vector Construction The rBMEV1 and rBMEV2 gene sequences designed in Example 1 were synthesized by a biotechnology company, and codon optimization was performed for the *E. coli* expression system. The optimized gene fragments were cloned into the prokaryotic expression vector pET-28a(+) using BamHI and XhoI restriction endonuclease sites, respectively, to construct recombinant plasmids pET-28a-rBMEV1 and pET-28a-rBMEV2. DNA sequencing verified that the sequences were consistent with the target sequences; the DNA sequence size of rBMEV1 was 1158 bp, and the DNA sequence size of rBMEV2 was 1767 bp. Figure 5 As shown.

[0066] 2.2 Protein-induced expression The recombinant plasmids that were correctly sequenced were transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and inoculated into LB liquid medium containing kanamycin (50 µg / mL) and cultured at 37°C with shaking until the OD600 was approximately 0.6-0.8. Isopropyl-β-D-thiogalactoside was added to a final concentration of 0.2 mM, and expression was induced at 25°C for 8-12 hours.

[0067] 2.3 Protein purification The bacterial cells were collected by centrifugation, sonicated, and then the supernatant (soluble fraction) was separated by centrifugation. Purification was performed using a Ni-NTA affinity chromatography column. The specific steps included: binding the protein supernatant containing the His tag to Ni-NTA resin, washing off contaminating proteins with a buffer containing 20 mM imidazole, and finally eluting the target protein with a buffer containing 250-500 mM imidazole.

[0068] The purified protein was dialyzed with PBS to replace the buffer. The protein concentration was determined using the BCA method, and the purity, molecular weight, and specificity of the protein were verified by SDS-PAGE and Western Blot (using an anti-His-tagged antibody).

[0069] The results showed that the protein bands of rBMEV1 and rBMEV2 were approximately 40 kDa and 63 kDa, respectively. Further validation using Western blot analysis with an anti-his-tagged antibody revealed specific immunoreaction bands of corresponding sizes. Figure 6 ).

[0070] Example 3: Immunogenicity and Safety Evaluation of the Vaccine 3.1 Animal Immunization Six- to eight-week-old female BALB / c mice were randomly divided into four groups (n=6): PBS negative control group, commercially available live attenuated vaccine S2 positive control group, rBMEV1 experimental group, and rBMEV2 experimental group.

[0071] Immunization regimen: Mice in each group were immunized via subcutaneous or intramuscular injection. Each mouse in the rBMEV1 and rBMEV2 experimental groups received 30 μg of the corresponding purified protein per injection, for a total of three immunizations, with each injection spaced two weeks apart. The negative and positive control groups were injected with PBS or S2 vaccine, respectively, according to the same regimen.

[0072] 3.2 Humoral immune response detection Blood was collected from the tail vein of mice before the first immunization (day 0) and one week after each immunization. Serum was separated and used to detect specific antibody responses, such as... Figure 7 As shown in Figure A.

[0073] Specific IgG antibody titer: Indirect ELISA method was used. The plate was coated with purified rBMEV1 or rBMEV2 protein. Serially diluted mouse serum was added to the wells, incubated, and then HRP-labeled goat anti-mouse IgG secondary antibody was added. After color development, the OD450 value was measured, and the antibody titer was calculated.

[0074] IgG subtype analysis: Using the same method as above, HRP-labeled goat anti-mouse IgG1 or IgG2a specific secondary antibodies were used to detect the antibody levels of IgG1 and IgG2a in serum to assess the bias of the immune response (Th1 / Th2 balance).

[0075] The results are as follows Figure 7 As shown, compared with the S2 vaccine and PBS control groups, the level of rBMEV1-specific IgG antibodies in the rBMEV1 vaccine continued to increase after booster immunization and remained elevated at 42 DPI. In contrast, there was no significant difference in IgG levels between the S2 group and the PBS group at 14 and 28 DPI. Figure 7 B). Further analysis of the IgG subclass profile showed that, compared with the S2 and PBS controls, the rBMEV1 vaccine induced significantly higher IgG1 and IgG2a titers. A sharp increase in IgG1 was observed at 42 DPI, while IgG2a levels began to rise significantly at 28 DPI. Figure 7 (C, D). Considering that the IgG2a / IgG1 ratio is often used as an indicator of potential Th1 or Th2 responses, this invention calculates the ratio of different IgG subtypes.

[0076] The results showed that rBMEV1 vaccination induced a systemic TH1 bias response, with an IgG2a / IgG1 ratio exceeding 1.0. Figure 7 E).

[0077] In mice immunized with rBMEV2, antigen-specific IgG responses were significantly detected at 28 DPI. Following booster immunization, titers were significantly increased at 28 and 42 DPI compared to the S2 protein group and the PBS control group, remaining at significantly higher levels. During the observation period of 14–42 DPI, IgG levels in the S2 group were not significantly different from those in the PBS control group. Figure 7 F). IgG subclass analysis showed that the rBMEV2 vaccine also increased the production of IgG1 and IgG2a by 28 DPI, with titers significantly exceeding those of the control group (F). Figure 7 G, H). However, in contrast to the TH1-biased response induced by rBMEV1, the immune response induced by rBMEV2 is biased towards the Th2 phenotype, as indicated by an IgG2a / IgG1 ratio below 1.0 (G, H). Figure 7 (I) This result demonstrates that the present invention, through differentiated structural design, successfully achieves targeted regulation of immune response types, providing two complementary candidate vaccine strategies for the prevention and control of brucellosis.

[0078] 3.3 Detection of cellular immune response One week after the last immunization, mice were sacrificed, and their spleens were aseptically harvested for cellular immunoassay. Figure 8 As shown in Figure A.

[0079] Splenic lymphocyte proliferation assay: Single-cell suspensions were prepared and cells were stimulated with ConA (positive control), culture medium (negative control), and 10 µg / mL rBMEV1 / rBMEV2 antigen, respectively. After culturing for 72 hours, cell proliferation was detected by CCK-8 assay and the stimulation index (SI) was calculated.

[0080] Flow cytometry analysis: Spleen cells were collected and stained with fluorescently labeled anti-mouse CD3, CD4, and CD8a antibodies, respectively, and then analyzed by flow cytometry. + T cells and CD8 + Changes in the proportion of T cells in lymphocytes.

[0081] Cytokine detection: The levels of Th1 / Th2 related cytokines such as IFN-γ, IL-2, IL-4, and IL-10 were measured by stimulating the culture supernatant of spleen cells with antigens or by directly detecting serum using a cytokine detection kit (CBA or ELISA).

[0082] Systematic evaluation revealed that both candidate vaccines rBMEV1 and rBMEV2 effectively elicited strong cellular immune responses, such as Figure 8As shown in Figure A. The experimental results showed that, at day 42 post-vaccination (42 DPI), the spleen-to-body weight ratio was significantly increased in all vaccination groups (S2, rBMEV1, and rBMEV2) compared to the PBS control group. At 42 DPI, the enhancement induced by rBMEV1 and rBMEV2 was more pronounced than that induced by S2, indicating robust and sustained systemic immune activation. Figure 8 B). At 42 DPI, the spleen lymphocyte proliferation stimulation index (SI) was significantly increased in all vaccine groups, with rBMEV1 and rBMEV2 eliciting a stronger proliferative response than S2. Figure 8 C).

[0083] To further characterize T lymphocyte responses, cytokine secretion profiles were analyzed using a double-antibody sandwich ELISA at 42 DPI. The levels of th1-type cytokines (IL-2, IFN-γ, and TNF-α) and th2-type cytokines (IL-4 and IL-10) in splenocytes were quantified. Figure 8 (DH). Compared with the PBS control group, all inoculation groups showed significantly increased secretion of these cytokines. Specifically, the rBMEV1 group showed the highest levels of IL-2, IFN-γ, and TNF-α among all groups. In contrast, the rBMEV2 group showed the highest levels of IL-4 and IL-10 at 42 DPI.

[0084] To elucidate the cellular immunity mechanisms mediated by the two candidate vaccines, spleen CD3 counts were assessed by flow cytometry at 42 DPI. + T cell proliferation and CD4+ + and CD8 + T cell subset differentiation. Compared with the PBS control group, inoculation with S2, rBMEV1, or rBMEV2 significantly enhanced CD3 differentiation. + CD4 + T cell activation was most significantly observed in the rBMEV2 group. Figure 8 I). In contrast, rBMEV1-induced CD3 + CD8 + T cell responses were stronger than those with rBMEV2, S2, or PBS. Figure 8 J).

[0085] These results demonstrate that the present invention achieves functional complementarity between the two vaccines at the cellular immune response level through structural differentiation design, providing a dual strategy for brucellosis covering Th1 / CTL and Th2 / Th cellular immunity.

[0086] 3.5 Safety Evaluation Clinical observation and weight monitoring: The mice's mental state, diet, and activity were observed daily, and their weight was measured twice a week. Figure 9 A).

[0087] Histopathological analysis: Two weeks after the last immunization, mice were sacrificed, and heart, liver, spleen, lung, kidney, and muscle tissue from the injection site were collected. These tissues were fixed in formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope for pathological changes such as inflammation, necrosis, and abnormal tissue structure. Figure 9 A).

[0088] Results: Throughout the trial, all vaccine-treated mice showed steady weight gain and no abnormal clinical manifestations. Figure 9 B). Histopathological sections showed that the structures of major organs were clear, and no pathological damage such as inflammatory cell infiltration or necrosis related to the vaccine was observed. Figure 9 C).

[0089] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Brucella recombinant multi-epitope vaccine protein, characterized in that, The vaccine protein is a first vaccine variant rBMEV1 or a second vaccine variant rBMEV2; the amino acid sequence of the first vaccine variant rBMEV1 is shown in SEQ ID NO.24; and the amino acid sequence of the second vaccine variant rBMEV2 is shown in SEQ ID NO.

25.

2. A pharmaceutical composition, characterized in that, It comprises the vaccine protein as described in claim 1, and a pharmaceutically acceptable carrier or adjuvant.

3. The use of the vaccine protein of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for the prevention of brucellosis.

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