Enhanced brucella outer membrane vesicle as well as preparation method and application thereof

Abstract

The invention provides an enhanced brucella outer membrane vesicle (OMV) as well as a preparation method and application thereof. The core innovation of the preparation method is as follows: besides conventional culture conditions, culture conditions for simulating an intracellular living environment are additionally introduced to culture a 104M: Omp19 overexpression strain, and then the enhanced OMV which is more comprehensive in antigen composition and can better simulate a real infection state is extracted from the 104M: Omp19 overexpression strain. The preventive brucella vaccine prepared on the basis of the method does not need to add any adjuvant, and can generate a relatively good protection effect through two-needle immunization at a low dose (5 mu g).

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an enhanced Brucella outer membrane vesicle, its preparation method, and its application. Background Technology

[0002] Brucella is an intracellular parasitic Gram-negative coccus, and brucellosis caused by it is classified as a Class B infectious disease in China, affecting both humans and animals. Statistics show that brucellosis is prevalent in more than 170 countries and regions worldwide. Vaccination is currently the primary means of controlling this disease. In the veterinary field, live attenuated vaccines such as swine S2 and sheep M5 have been widely used and have played a crucial role in controlling brucellosis in animals. Regarding human vaccines, apart from the 104M scratch-skin human brucellosis live vaccine approved for use in my country, no other commercially available brucellosis vaccines have been approved for marketing internationally. Although the 104M live vaccine has good immunoprotective efficacy, its residual virulence leads to significant adverse reactions, making it unsuitable for large-scale vaccination. Furthermore, its scratch-skin injection method easily causes local pain, resulting in low public acceptance; therefore, it is currently mainly used as a strategic reserve vaccine.

[0003] Outer membrane vesicles (OMVs) are spherical, nanoscale vesicle structures secreted by Gram-negative bacteria. Due to their rich content of various functional proteins and advantages such as non-replicative ability, non-infectiousness, and the ability to activate a relatively strong immune response without adjuvants, they hold great potential in vaccine development. Patent CN113025640A discloses a method for preparing Brucella outer membrane vesicles and their applications. Brucella bacteria are obtained by modifying the bacteria using genetic engineering methods, targeting one or more of OMP39, BamD, OMP2b, Cgs, and BF3285c. hb20 Five gene-deleted mutant strains were successfully obtained. The results of culturing and extracting outer membrane vesicles from these strains showed a significant increase in the yield of outer membrane vesicles in three of the modified strains. However, this technical solution has the following key technical defects: 1) The OMV in this patent requires a high immunization dose, combined with aluminum adjuvant, and two subcutaneous immunizations. Furthermore, under the condition of "30 CFU / animal low-dose Brucella M28 strain challenge," the highest protection rate is only 80%. Compared with the clinical protection requirements of brucellosis vaccines, its protective efficacy is still low and cannot meet the immunization protection requirements of high-risk groups such as herders and veterinarians; 2) OMV is derived from self-isolated Brucella. hb20 However, the toxicity, immunogenicity, and safety of this strain have not been systematically verified, and it does not yet have the basis for application as the original strain for human OMV vaccines, thus failing to guarantee the clinical safety and immunogenicity of subsequent OMV vaccines.

[0004] In summary, existing human brucellosis vaccines have problems in two aspects: safety (residual virulence and adverse reactions) and vaccination compliance (painful administration method and low acceptance). There is an urgent need to develop new human brucellosis vaccines with good safety, high protective efficacy and convenient administration. Summary of the Invention

[0005] The purpose of this invention is to solve the technical problems of existing Brucella vaccines having low protective efficacy or unreliable safety.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides a method for preparing the enhanced Brucella outer membrane vesicles described above, comprising the following steps: Brucella 104M:Omp19 overexpression strain was cultured under normal culture conditions and / or simulated intracellular survival conditions. The culture was then subjected to differential centrifugation and filtration to obtain the supernatant. The supernatant was subjected to ultracentrifugation, and the precipitate was collected to obtain the enhanced Brucella outer membrane vesicles.

[0007] Preferably, the conventional culture conditions are cultured in TSB medium for 48-72 hours.

[0008] Preferably, the simulated intracellular survival conditions include: First, pre-culture in TSB medium until the logarithmic growth phase; Transfer to GEM nutrient deficiency medium and continue culturing for 24-48 hours.

[0009] The present invention also provides an enhanced Brucella outer membrane vesicle prepared by the preparation method described above.

[0010] The present invention also provides the application of the enhanced Brucella outer membrane vesicles described above in the preparation of a vaccine for the prevention of brucellosis.

[0011] The present invention also provides a vaccine for the prevention of brucellosis, comprising enhanced brucellosis outer membrane vesicles prepared by the preparation method described above.

[0012] Preferably, the vaccine does not contain exogenous adjuvants. Existing technology CN113025640A uses an hb20 gene deletion strain with unclear safety and immunoprotective efficacy, and prepares OMV under conventional culture conditions, resulting in limited protective efficacy and reliance on adjuvants and high-dose immunization. This invention, by employing a 104M:Omp19 overexpression strain with confirmed immunoprotective efficacy and simulating intracellular environmental stimulation, significantly broadens the antigen spectrum and enhances immunogenicity, thereby achieving highly efficient protection under adjuvant-free and low-dose conditions.

[0013] Preferably, the immunization schedule for the vaccine is two doses, with a two-week interval between doses.

[0014] Preferably, the vaccine is administered via intramuscular injection.

[0015] The present invention also provides a method for preventing brucellosis, comprising administering an effective amount of the enhanced brucellosis outer membrane vesicles to an individual in need of such treatment.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a method for preparing enhanced Brucella outer membrane vesicles (OMV). The method is characterized by innovatively introducing culture conditions that simulate the intracellular environment of the host cell, based on conventional culture, to induce the culture of the Brucella 104M:Omp19 overexpressing strain, thereby extracting an "enhanced OMV" with a more comprehensive antigenic composition and closer to the actual infection state. This optimized culture strategy can effectively induce the expression of stress proteins and virulence factors that play a key role in the natural infection process, thereby significantly broadening the antigenic spectrum carried by the OMV, enhancing the recognition and response strength of the immune system, and ultimately improving the overall protective efficacy of the vaccine. This successfully solves the key technical problem of insufficient immunogenicity in existing OMV vaccines. Furthermore, since OMV itself does not possess the ability to replicate as a complete bacterium, there is no risk of inducing persistent infection or virulence recovery. Therefore, the enhanced OMV prepared by this invention has better safety compared to its parent strain 104M and the attenuated live vaccine based on this strain.

[0017] The enhanced Brucella outer membrane vesicle (OMV) provided by this invention is derived from the Brucella 104M:Omp19 overexpression strain. This strain, while retaining the known protective efficacy and human safety of the 104M strain, significantly increases the load of protective antigen through Omp19 overexpression, thus laying the core material basis for the preparation of a high-protective OMV. This contrasts with insufficiently validated... hb20 Compared to wild strains, the present invention has a clear safety profile and superior protective effect; compared to wild strains, it completely avoids the risk of toxicity reversion while ensuring the protective effect.

[0018] The brucellosis prevention vaccine provided by this invention achieves a high level of protective efficacy with only two low-dose (5μg) immunizations without the addition of any adjuvants. The vaccine fully utilizes the high immunogenicity and strong protective efficacy of the OMV formulation of this invention, significantly optimizing the vaccination experience and overall safety while avoiding potential adverse reactions from adjuvants, thus possessing superior clinical applicability and promising prospects for widespread application. Attached Figure Description

[0019] Figure 1Electron micrographs of 104M:Omp19 TOMV (A) and 104M:Omp19 GOMV (B) of Embodiment 1 of the present invention; Figure 2 This is a statistical chart of spleen weight in mice immunized with 5 μg OMV and in the control group 7 days after challenge with the virus in Example 2 of the present invention. Spleen weight: spleen weight; Figure 3 This is a statistical graph of bacterial load in the spleen of mice immunized with 5 μg OMV and in the control group 7 days after challenge. Log CFUs / Spleen: the logarithm of the number of bacteria per spleen. Figure 4 This is a blood routine analysis image of mice 28 days after immunization with 10 μg OMV according to Example 2 of the present invention. In the image, top left: white blood cell count (WBC); top right: absolute lymphocyte count (LYM#); bottom left: absolute granulocyte count (GRA#); bottom right: platelet count (PLT). Figure 5 This is a blood biochemical analysis diagram of mice 28 days after immunization with 10 μg OMV according to Example 2 of the present invention. In the diagram, top left: alanine aminotransferase (ALT); top right: aspartate aminotransferase (AST); bottom left: creatinine (CR); bottom right: blood urea nitrogen (BUN). Detailed Implementation The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0020] Example 1 Preparation and characterization of 104M:Omp19 TOMV and 104M:Omp19 GOMV 1. Preparation of outer membrane vesicle supernatant (hereinafter referred to as 104M:Omp19 TOMV) from Brucella TSB culture supernatant: Take 100 μL of Brucella 104M:Omp19 overexpression strain cryopreservation solution and inoculate it into 10 mL TSB at a ratio of 1:100. Incubate at 37℃ and 220 rpm until the peak logarithmic growth phase (16-24 hours). Transfer the above bacterial culture to 1 L TSB medium and incubate at 37℃ and 220 rpm for 48-72 h. Centrifuge the bacterial culture at 4000 g for 15 min. Discard the precipitate and repeat the above centrifugation process. Transfer the supernatant to a 0.22 μm vacuum filter cup and filter twice to further remove residual bacterial cells and large-particle impurities. Collect the filtered supernatant and use it for subsequent experiments after sterility verification. The Brucella 104M:Omp19 overexpression strain was designed and constructed by our research group. For the specific construction process, please refer to the published literature: Yang Qiaoling, Zai Xiaodong, Yin Ying, et al. Construction and immunoprotective evaluation of Brucella 104M:Omp19 overexpression strain [J]. Biotechnology Communications, 2018, (1): 1-6.

[0021] 2. Outer membrane vesicles in Brucella GEM culture supernatant (hereinafter referred to as 104M:Omp19 GOMV): Take 100 μL of Brucella 104M:Omp19 overexpression strain cryopreservation solution and inoculate it into 10 mL TSB at a ratio of 1:100. Incubate at 37℃ and 220 rpm until the peak logarithmic growth phase (16-24 hours). Transfer the above bacterial solution to 1 L TSB medium and incubate at 37℃ and 220 rpm for 24 h. Centrifuge the bacterial solution at 4000 g for 15 min. Discard the supernatant, resuspend the precipitate in 1 L GEM auxotrophic medium, and continue to incubate at 37℃ and 220 rpm for 24-48 h. Centrifuge the bacterial solution at 4000 g for 15 min. Discard the precipitate and repeat the above centrifugation process. Transfer the supernatant to a 0.22 μm vacuum filter cup and filter twice. Collect the filtered supernatant and use it for subsequent experiments after sterility verification.

[0022] The GEM medium formula is as follows: glucose 20 g / L, K₂HPO₄ 10 g / L, citric acid 2 g / L, MgSO₄•7H₂O 0.2 g / L, NaNH₄HPO₄•4H₂O 3.5 g / L, autoclaved at 121℃ for 15 min. This medium is an auxotrophic medium, which can simulate the nutrient restriction conditions experienced by Brucella bacteria within macrophages after infecting the human body.

[0023] 3. OMV Extraction: OMV was extracted using ultracentrifugation. The sterile filtered supernatant was ultrafiltered through a 100 kDa membrane or ultrafiltration tube and transferred to an ultracentrifuge tube. Centrifugation was performed at 110,000 g, 4°C, for 2 h. The precipitate was washed once with pre-chilled sterile PBS and then resuspended in 1 mL of sterile PBS. This precipitate was the enriched OMV. The total protein concentration was determined using a BCA protein assay kit. After filtration through a 0.22 μm syringe filter, the precipitate was aliquoted into sterile EP tubes and stored at -80°C for long-term preservation.

[0024] 4. OMV Characterization: The morphology and particle size distribution of the two OMVs were further observed using transmission electron microscopy, such as... Figure 1 The electron microscopy morphology of 104M:Omp19 TOMV and 104M:Omp19 GOMV were shown. The results showed that the OMV vesicles derived from Brucella 104M:Omp19 had clear boundaries and complete membrane structures.

[0025] Example 2 1. Mouse immunization program and challenge protection experiment Experimental subjects: SPF-grade BALB / c mice (6-8 weeks old, female), 5 mice per group; Experimental group 1: 104M:Omp19 TOMV and 104M:Omp19 GOMV prepared in Example 1, 5 μg / animal, intramuscular injection, 2 immunizations, 2 weeks apart, without adjuvant; Control group: PBS (phosphate-buffered saline), immunized twice, 2 weeks apart, without adjuvant, volume the same as the experimental group; Challenge conditions: Intraperitoneal injection of Brucella bovis strain A19 (1×10⁻⁶) 6 CFU / each); Detection indicators: Spleen weight and spleen bacterial load (Log) were measured 7 days after viral challenge. 10 CFU / spleen); Experimental Procedure: Mice were immunized with two types of OMV using a two-dose intramuscular immunization regimen on days 0 and 14. These mice were then challenged intraperitoneally with Brucella bovine strain A19 at a dose of 10... 6 CFU / mouse. Seven days after challenge, mice were sacrificed, and their spleens were weighed and the bacterial load was measured.

[0026] like Figure 2The spleen weights of mice immunized with 5 μg of OMV and in the control group were measured 7 days after challenge. The mean ± standard deviation of spleen weight data after challenge were as follows: PBS negative control group (0.313 ± 0.115 g), 104M:Omp19 TOMV 5 μg group (0.132 ± 0.022 g), and 104M:Omp19 GOMV 5 μg group (0.127 ± 0.021 g). Statistical analysis showed significant differences between the two OMV immunization groups and the PBS control group. P The value <0.01 indicates that the OMV vaccine can significantly reduce the inflammatory response of the spleen caused by infection. It is worth noting that there was no statistically significant difference in spleen weight between the TOMV and GOMV groups, and both were similar to normal mice of the same age, indicating that the two OMV vaccines have comparable effects in reducing pathological reactions and have good protective effects.

[0027] like Figure 3 The results show the bacterial load in the spleen of mice 7 days after challenge with the 5 μg immunization group and the control group. Data showed that, without any exogenous adjuvants, the protective efficacy induced by both 5 μg doses of enhanced OMV was significantly better than that of the PBS negative control group, indicating that both adjuvant-free enhanced OMVs could induce good immunoprotective efficacy at this dose. Compared with the PBS control group, the bacterial load in the spleen of the GOMV and TOMV immunization groups decreased by approximately 216-fold and 110-fold, respectively, with the bacterial load in the TOMV group being twice that of the GOMV group, suggesting that GOMV has a superior protective effect. This indicates that culture conditions mimicking the intracellular environment of the host cell can effectively induce bacteria to express stress proteins and virulence factors that play a key role in natural infection, thereby enhancing the immune system's recognition and response, and ultimately improving the overall protective efficacy of the vaccine.

[0028] Spleen bacterial load in each group was expressed as Log 10 The mean ± standard deviation (CFU) values ​​are as follows: PBS negative control group: 7.354 ± 0.405; 104M:Omp19 TOMV 5 μg group: 5.064 ± 0.606; 104M:Omp19 GOMV 5 μg group: 4.868 ± 0.540. The protection index was calculated using the formula (PBS group Log...). 10 Mean - Vaccine Group Log 10The mean values ​​were 2.29 and 2.49 for the 104M:Omp19TOMV and GOMV groups, respectively. Referring to standard criteria for evaluating Brucella vaccine efficacy, a protection index ≥2.0 is considered a critical threshold for vaccine effectiveness. Our results show that low-dose (5 μg) doses of both enhanced OMV formulations significantly reduced bacterial load in the spleen of mice after challenge, and both had protection indices above 2.0, indicating that 104M:Omp19TOMV and GOMV can still induce good immunoprotective effects at low doses and without adjuvants.

[0029] In summary, in the embodiments of the present invention, mice immunized with enhanced OMV showed significantly lower bacterial load in their spleens after challenge compared to the PBS control group. This contrasts with existing technologies (such as CN113025640A). hb20 The immunogenicity and protective efficacy of gene-deleted strains are not yet clear, and effective protection requires adjuvant assistance and high-dose immunization. Furthermore, hb20 The virulence background of existing strains lacks systematic research, and their preparation process and potential clinical applications pose certain safety risks. In contrast, this invention uses an immunogenic 104M:Omp19 overexpression strain as the production strain and innovatively combines stimulation with culture conditions simulating the intracellular environment, significantly broadening the antigenic spectrum of OMV and comprehensively enhancing its immunogenicity. Therefore, this invention can achieve highly effective protection without adjuvants and with only low-dose immunization, effectively overcoming the technical bottleneck of the prior art where efficacy and safety are difficult to balance.

[0030] 2. OMV Safety Evaluation The initial safety evaluation of the OMV vaccine was conducted by detecting blood routine and blood biochemical indicators in immunized mice.

[0031] Experimental subjects: SPF grade BALB / c mice (6-8 weeks old, female), 6 mice per group; Experimental group: 10 μg / animal of 104M:Omp19 TOMV and 104M:Omp19 GOMV prepared in Example 1, immunized twice by intramuscular injection, with an interval of 2 weeks; Control group: PBS (same inoculation method and frequency as experimental group); Testing indicators: Blood was collected on day 28 after the first immunization for blood biochemistry and routine blood analysis; The experiment used a two-dose immunization regimen of 10 μg OMV. Blood samples were collected from mice 28 days post-immunization for quantitative determination and analysis of key blood routine and blood biochemical indicators. Figure 4The study analyzed the complete blood count of mice 28 days after immunization with 10 μg OMV. The results showed that although there were slight fluctuations in the blood count indicators (including white blood cell count (WBC), absolute lymphocyte count (LYM#), absolute granulocyte count (GRA#), and platelet count (PLT)) of the two OMV immunization groups, all indicators were within the normal physiological range and there were no statistically significant differences compared with the blank control group.

[0032] like Figure 5 The study analyzed the blood biochemistry of mice 28 days after immunization with 10 μg OMV. The results showed that, among the blood biochemical indicators, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which reflect liver function, and blood urea nitrogen (BUN) and creatinine (CR), which reflect kidney function, no significant differences were observed between the two OMV immunization groups and the PBS control group, with only slight fluctuations in a few indicators.

[0033] In summary, all blood biochemical and routine blood indicators in the two enhanced OMV immunization groups showed no statistically significant differences compared to the PBS control group, indicating that the OMV of this invention has good safety. The 104M attenuated live vaccine, due to the use of live bacteria, carries inherent residual virulence and the risk of virulence reversion, thus its safety is insufficient. This invention uses OMV secreted by the 104M:Omp19 overexpression strain as the vaccine antigen, which is an inactive bacterial metabolite, fundamentally avoiding the safety risks associated with the use of live bacteria.

[0034] Regarding vaccination strategies and safety, existing methods carry varying degrees of risk: on the one hand, the 104M live vaccine requires a painful skin-scarring method, and the resulting local and systemic adverse events are primarily due to the inherent toxicity of the live vaccine itself; on the other hand, existing OMV vaccines (such as CN113025640A) rely on adjuvants and have high immunization doses, which also easily trigger local reactions and increase the risk of systemic adverse reactions. In contrast, the OMV formulation provided by this invention is an adjuvant-free, inactive vaccine component, administered via a minimally painful, standardized intramuscular injection method. This strategy fundamentally avoids the risks of adverse reactions caused by the toxicity of live vaccines or adjuvants, thereby significantly improving the safety and compliance of clinical use while ensuring effective immunization.

[0035] In this embodiment of the invention, to provide clear and reproducible experimental data, the protocol and results of immunization via intramuscular injection are specifically described and verified. However, this description is merely illustrative and is not intended to limit the invention. Those skilled in the art will understand that the physicochemical properties of the enhanced Brucella outer membrane vesicles make them equally suitable for effective immunization via subcutaneous injection, intradermal injection, mucosal immunization (including but not limited to nasal inhalation or oral administration), and other feasible non-intramuscular injection routes. Based on the insights gained from the technical solutions of this invention, these alternative routes can all elicit effective protective immune responses through conventional dose optimization and immunization procedure adjustments, and therefore should all be considered to fall within the scope of protection of this invention.

[0036] The above are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of the claims of the present invention pending approval.

Claims

1. A method for preparing enhanced Brucella outer membrane vesicles, characterized in that, Includes the following steps: Brucella 104M:Omp19 overexpression strain was cultured under normal culture conditions and / or simulated intracellular survival conditions. The culture was then subjected to differential centrifugation and filtration to obtain the supernatant. The supernatant was subjected to ultracentrifugation, and the precipitate was collected to obtain the enhanced Brucella outer membrane vesicles.

2. The method according to claim 1, characterized in that, The standard culture conditions are as follows: culture in TSB medium for 48-72 hours.

3. The method according to claim 1, characterized in that, The simulated intracellular survival conditions include: First, pre-culture in TSB medium until the logarithmic growth phase; Transfer to GEM nutrient deficiency medium and continue culturing for 24-48 hours.

4. An enhanced Brucella outer membrane vesicle prepared by the preparation method according to any one of claims 1 to 3.

5. The use of the enhanced Brucella outer membrane vesicles as described in claim 4 in a vaccine for the prevention of brucellosis.

6. A vaccine for the prevention of brucellosis, characterized in that, Includes the enhanced Brucella outer membrane vesicles as described in claim 4.

7. The vaccine according to claim 6, characterized in that, The vaccine does not contain exogenous adjuvants.

8. The vaccine according to claim 7, characterized in that, The immunization schedule for the vaccine is two doses, two weeks apart.

9. The vaccine according to claim 7, characterized in that, The vaccine is administered via intramuscular injection.

10. A method for preventing brucellosis, characterized in that, This includes administering an effective amount of the enhanced Brucella outer membrane vesicles as described in claim 4 to individuals in need.

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