Multi-drug-resistant acinetobacter baumannii recombinant vesicular subunit vaccine and application thereof

Abstract

The invention provides a multi-drug-resistant acinetobacter baumannii recombinant vesicular subunit vaccine and application thereof. The subunit vaccine takes an acinetobacter baumannii bilayer membrane vesicle (BBV) as a delivery carrier and a self-adjuvant; the surface of the double-layer membrane vesicle of the acinetobacter baumannii is loaded with three antigen components: an outer membrane protein A (OmpA) of the acinetobacter baumannii, an outer membrane protein W (OmpW) of the acinetobacter baumannii and an outer membrane protein 22 (Omp22) of the acinetobacter baumannii. According to the invention, the limitation of the traditional single target vaccine is broken through, the BBV is used for providing a membrane-related antigen, three core outer membrane proteins of OmpA, Omp22 and OmpW are combined, and pathogenic key links such as multi-dimensional targeting bacterial adhesion, biomembrane formation, drug resistance and the like are realized. On the basis of the natural strain adaptability of the BBV and the high conservative property of the three proteins, the antibody generated through induction after vaccine immunization can be subjected to cross recognition with at least 10 different serotype multi-drug-resistant strains, the protection range is greatly widened, and the problem that an existing vaccine is prone to failure due to antigen variation is solved.

Description

Technical Field

[0001] This invention belongs to the field of vaccine preparation technology, and specifically relates to a recombinant vesicular subunit vaccine of multidrug-resistant Acinetobacter baumannii and its application. Background Technology

[0002] Acinetobacter baumannii is a Gram-negative bacterium and an important opportunistic pathogen in hospitals. Due to the increasing number of multidrug-resistant strains and its global prevalence, Acinetobacter baumannii infection has become a challenging problem in clinical practice. In light of this, the U.S. Centers for Disease Control and Prevention (CDC) has listed multidrug-resistant Acinetobacter baumannii as a serious threat, prompting ongoing public health surveillance and prevention activities. Furthermore, the World Health Organization (WHO) has included carbapenem-resistant Acinetobacter baumannii in its list of key bacteria posing the greatest threat to human health.

[0003] Acinetobacter baumannii infections in hospitals are mostly multidrug-resistant bacteria with large genomes, complex pathogenic mechanisms, and numerous targets. The key to preventing infection lies in accurately identifying antigen targets and developing vaccines that can efficiently induce an immune response in the body.

[0004] Vaccines are an effective tool for preventing and controlling Acinetobacter baumannii infection. Many researchers are investigating subunit vaccines against Acinetobacter baumannii. Subunit vaccines have advantages such as high purity, safety and stability, ease of production, and strong specificity in inducing immune responses. To date, no Acinetobacter baumannii subunit vaccine candidates have entered clinical trials. This may be related to the fact that subunit vaccines are easily degraded in vivo and have weak immunogenicity. Current vaccine development for Acinetobacter baumannii is mainly based on single-target subunit vaccines, which are difficult to provide multi-target, comprehensive protective effects in the real world.

[0005] The Acinetobacter baumannii genome is large and complex, but existing technologies target only a single site and fail to provide broad-spectrum protection. Several key obstacles remain to be overcome in the development of existing Acinetobacter baumannii vaccines, including improving vaccine stability, immunogenicity, breadth and consistency of protection, and addressing regulatory compliance issues. These are detailed below: (1) Antigenic variability: Acinetobacter baumannii antigens are variable, which may result in subunit vaccines not providing broad protection.

[0006] (2) Adjuvants or delivery systems are required: Many subunit vaccine candidates require the addition of adjuvants or delivery systems to enhance their stability and immunogenicity.

[0007] (3) Limitations of protection: Some vaccine candidates may only provide protection against specific serotypes of Acinetobacter baumannii, rather than broad protection.

[0008] (4) Variability of vaccine efficacy: In terms of preclinical survival, different subunit vaccine candidates showed a wide range of efficacy from 14% to 100%, which indicates that vaccine efficacy is highly variable.

[0009] Therefore, developing a multi-target, highly immunogenic, safe, stable, and broad-spectrum protection Acinetobacter baumannii vaccine is an urgent problem to be solved in the clinical prevention or treatment of multidrug-resistant Acinetobacter baumannii infection. Summary of the Invention

[0010] To address the problems existing in the prior art, the purpose of this invention is to design a multi-target, highly immunogenic, safe, stable, and broad-spectrum Acinetobacter baumannii vaccine, and to provide a recombinant vesicular subunit vaccine for multidrug-resistant Acinetobacter baumannii and its application.

[0011] The objective of this invention is achieved through the following technical solution: A first aspect of the present invention provides a recombinant vesicular subunit vaccine against multidrug-resistant Acinetobacter baumannii, wherein the subunit vaccine uses Acinetobacter baumannii bilayer membrane vesicles (BBV) as a delivery carrier and self-adjuvant, and the surface of the Acinetobacter baumannii bilayer membrane vesicles is loaded with at least three antigenic components, including Acinetobacter baumannii outer membrane protein A (OmpA), Acinetobacter baumannii outer membrane protein W (OmpW), and Acinetobacter baumannii outer membrane protein 22 (Omp22).

[0012] Furthermore, the gene sequence of OmpA is shown in SEQ ID NO.1: Acinetobacter baumannii outer membrane protein A (OmpA) plays a crucial role in the bacterium's physiological functions, pathogenic mechanisms, and interactions with its host. The main functions of OmpA are as follows: As a channel protein: OmpA is the most abundant porin protein in the outer membrane of Acinetobacter baumannii. It acts as a small molecular channel, controlling the inflow and outflow of substances.

[0013] Antibiotic and serum resistance: OmpA is involved in the resistance of Acinetobacter baumannii to antibiotics and serum. The presence of OmpA helps the bacteria resist the host's immune defenses, including attacks from the complement system.

[0014] Biofilm formation: OmpA plays a key role in the formation of biofilms in Acinetobacter baumannii, helping the bacteria to colonize and survive on environmental surfaces.

[0015] Host-cell interaction: OmpA is involved in the adhesion and invasion of Acinetobacter baumannii with host cells. The lower lung lesions and blood bacterial load in a mouse pneumonia model by OmpA mutant strains indicate the importance of OmpA in bacterial pathogenicity.

[0016] Promoting apoptosis: OmpA can be released through outer membrane vesicles (OMVs) and target the mitochondria of host cells, leading to mitochondrial fragmentation and apoptosis.

[0017] Immune response: OmpA is a highly immunogenic protein that can serve as a target for both innate and adaptive immune responses in the host, leading to the clearance of bacteria.

[0018] Virulence factor: OmpA is a key virulence factor of Acinetobacter baumannii, and its overexpression is an independent risk factor for mortality from hospital-acquired pneumonia and bacteremia.

[0019] As a therapeutic target: Due to its key role in the pathogenesis of Acinetobacter baumannii, OmpA is considered a potential therapeutic target.

[0020] Regulation of host cell function: OmpA can increase the production of pro-inflammatory cytokines and induce lung epithelial barrier dysfunction, promoting bacterial translocation.

[0021] In summary, OmpA plays a multifaceted role in the pathogenic mechanism of Acinetobacter baumannii, including promoting bacterial adhesion, invasion, biofilm formation, drug resistance, and modulating the host's immune response. These functions make OmpA an important target for the research and treatment of Acinetobacter baumannii infection.

[0022] The gene sequence of OmpW is shown in SEQ ID NO.2: ; Acinetobacter baumannii outer membrane protein W (OmpW) is an outer membrane protein with the following functions and effects: Involved in biofilm formation: OmpW has been shown to promote biofilm formation in Acinetobacter baumannii, which is one of the important mechanisms of multidrug resistance and pathogenicity in Acinetobacter baumannii.

[0023] Substance transport: OmpW is an 8-chain β-barrel porin with long and narrow hydrophobic channels. It has been shown to participate in the transport of various cations, hydrophobic compounds, hypochlorous acid and other harmful substances, playing an important role in cell survival.

[0024] Iron transport: OmpW participates in the transport of iron ions, and the expression of OmpW is positively regulated by iron ions and iron transporter (fur), affecting the iron balance in the host.

[0025] Promoting host cell apoptosis: OmpW can induce host cell apoptosis in vitro, and may promote host cell apoptosis through some mechanism, thereby promoting the invasion of Acinetobacter baumannii.

[0026] Resistance to host phagocytosis: Studies have shown that OmpW proteins protect bacteria from host phagocytosis and are associated with bacterial virulence and pathogenicity.

[0027] Drug resistance-related: OmpW is associated with bacterial drug resistance and may lead to widespread drug resistance by mediating the efflux of certain drugs.

[0028] Immunogenicity: OmpW is immunogenic and can serve as a potential target for vaccine development.

[0029] In summary, OmpW plays multiple roles in the pathogenic mechanism of Acinetobacter baumannii, including promoting biofilm formation, participating in substance transport and iron ion transport, inducing host cell apoptosis, resisting host phagocytosis, and being associated with drug resistance. These functions make OmpW an important target for the research and treatment of Acinetobacter baumannii infection.

[0030] The gene sequence of Omp22 is shown in SEQ ID NO.3: .

[0031] Acinetobacter baumannii outer membrane protein 22 (Omp22) is an outer membrane protein that plays an important role in bacterial pathogenicity and immune responses. The main functions and characteristics of Omp22 are as follows: Immunogenicity: Omp22 is immunogenic and can induce the host to produce specific antibodies. Studies have shown that immunization with Omp22 can stimulate mice to produce an antibody response against Omp22.

[0032] Protective Immunity: Omp22 can induce a protective immune response. In mouse models, immunization with Omp22 significantly improved survival rates in mice facing lethal challenges from Acinetobacter baumannii, reduced bacterial burden in the lungs, spleen, liver, kidneys, and blood, and suppressed serum levels of inflammatory cytokines.

[0033] In vitro bactericidal activity: The Omp22 specific antibody has bactericidal activity against Acinetobacter baumannii in vitro, and this activity is partly dependent on the specific antibody.

[0034] Passive immune protection: Passive immunization experiments showed that the use of Omp22-OMV (outer membrane vesicle) antiserum can improve the survival rate of mice in the lethal challenge of Acinetobacter baumannii.

[0035] Cytotoxicity: Omp22 exhibits low cytotoxicity to mammalian cells. In in vitro experiments, Omp22 showed only a slight inhibitory effect on the proliferation of tumor cells A549 and normal cells 293FT, suggesting that Omp22 may be a safe vaccine candidate.

[0036] Acute toxicity test: Acute toxicity test in mice showed that high doses of Omp22 did not cause significant pathological changes, and all mice that received Omp22 survived.

[0037] Immunoprotective studies: Compared with other recombinant proteins, the Omp22 fusion protein OmpK / Omp22 showed stronger immunogenicity and immunoprotective properties.

[0038] In summary, Omp22, as an outer membrane protein, not only possesses immunogenicity but also provides protective immunity, reduces bacterial burden, and exhibits bactericidal activity in vitro. These properties suggest that Omp22 is a promising vaccine candidate for the prevention of Acinetobacter baumannii infection.

[0039] Furthermore, the mass ratio of BBV to OmpA, Omp22, and OmpW is 1-100 μg BBV + 1-100 μg OmpA + 1-100 μg OmpW + 1-100 μg Omp22; preferably, the mass ratio is 5 μg BBV + 50 μg OmpA + 15 μg OmpW + 10 μg Omp22.

[0040] The second aspect of this invention provides a method for preparing the recombinant vesicular subunit vaccine of multidrug-resistant Acinetobacter baumannii described in the first aspect, comprising the following steps: (1) Preparation of BBV: Multidrug-resistant Acinetobacter baumannii was cultured in a culture medium at 37°C with shaking. EDTA-2Na was added and the culture was continued with shaking. The bacterial precipitate was obtained by centrifugation. The bacterial precipitate was resuspended in an isotonic solution (such as HBSS, PBS, or physiological saline). The bacterial suspension was treated by a high-pressure homogenizer. The resulting suspension was centrifuged at 6000 rpm at 4°C. The supernatant was collected and centrifuged again at 100000 rpm at 4°C. The precipitate was resuspended in an isotonic solution (such as HBSS, PBS, or physiological saline). The precipitate was filtered to obtain Acinetobacter baumannii double membrane vesicle BBV. (2) Preparation of antigens: The encoding genes of OmpA, OmpW and Omp22 were inserted into prokaryotic expression plasmids to construct recombinant plasmids and transform competent cells (such as DH5α and BL21(DE3)). After expression induced by IPTG, OmpA, OmpW and Omp22 antigens with a purity of ≥90% were obtained by ultrasonic lysis and nickel ion affinity chromatography. (3) Antigen loading: BBV is mixed with three antigens, OmpA, OmpW and Omp22, according to the set mass ratio and incubated at 4℃-37℃ to obtain a mixture; (4) Ultrasonic treatment: The mixture is ultrasonically treated on ice with an ultrasonic power of 10-30%, each ultrasonic treatment lasts 5-30 minutes, and a total of 2-4 cycles are performed. (5) Extrusion purification: The ultrasonically purified mixture is extruded for 3-10 cycles through an extruder or through a high-pressure homogenizer to load the three antigens OmpA, OmpW, and Omp22 onto the Acinetobacter baumannii double membrane vesicles BBV. After purification, the multidrug-resistant Acinetobacter baumannii recombinant vesicle subunit vaccine is obtained.

[0041] Furthermore, the multidrug-resistant Acinetobacter baumannii recombinant vesicular subunit vaccine described in the first aspect can also be prepared by loading outer membrane protein antigens onto bacterial membrane vesicles in a genetically engineered, physical, or chemical manner.

[0042] The third aspect of this invention provides the use of the recombinant vesicular subunit vaccine for multidrug-resistant Acinetobacter baumannii described in the first aspect in the preparation of drugs for the prevention and / or treatment of Acinetobacter baumannii infection.

[0043] Furthermore, the drug may also include pharmaceutically acceptable carriers or excipients.

[0044] The advantages of this invention compared to the prior art are as follows: 1. The recombinant vesicular subunit vaccine against multidrug-resistant Acinetobacter baumannii described in this invention overcomes the limitations of traditional single-target vaccines. It utilizes Acinetobacter baumannii double-membrane vesicles (BBV) to provide membrane-associated antigens, combined with three core outer membrane proteins—OmpA, Omp22, and OmpW—to target key pathogenic factors such as bacterial adhesion, biofilm formation, and drug resistance from multiple dimensions. Leveraging the natural strain compatibility of BBV and the high conservation of the three proteins, the vaccine can cross-recognize at least three different serotypes of multidrug-resistant strains, significantly broadening the scope of protection and solving the problem of existing vaccines becoming ineffective due to antigen mutations. 2. In the multidrug-resistant Acinetobacter baumannii recombinant vesicular subunit vaccine of this invention, Acinetobacter baumannii double-membrane vesicles (BBV) function as both nanodelivery carriers and self-adjuvants. Their nanoparticle size (30-300nm) facilitates phagocytosis by dendritic cells, promoting antigen presentation. The lipopolysaccharide and other components they contain can activate innate immunity, enhancing the response without the need for exogenous adjuvants. After immunization, the antigen-specific antibody titer increases 2-3 times compared to the single antigen group, and it can also induce Th1 / Th2 / Th17 multi-type immune responses. The protection rate against mouse challenge reaches 100%, overcoming the shortcomings of existing subunit vaccines that are "weak immunogenicity and dependent on exogenous adjuvants." 3. The multidrug-resistant Acinetobacter baumannii recombinant vesicular subunit vaccine of the present invention adopts the form of "vesicular subunit", which eliminates the risk of virulence factors of whole bacterial vaccines and avoids the defects of toxicity risk of whole bacterial vaccines. Attached Figure Description

[0045] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 The preparation and characterization of the VAW22 (UE) vaccine are shown; Figure 2 The immunogenicity of the recombinant vesicular subunit vaccine of Acinetobacter baumannii and the cross-recognition characteristics of the antibodies induced by it with clinically isolated multidrug-resistant Acinetobacter baumannii are shown. Figure 3 This demonstrates the immune bias of Acinetobacter baumannii recombinant vesicular subunit vaccines; Figure 4 , Figure 5 The results show that immunization with the recombinant vesicular subunit vaccine of Acinetobacter baumannii can protect mice against clinically isolated lethal doses of multidrug-resistant Acinetobacter baumannii. Figure 6 The results show that immunization with the recombinant vesicular subunit vaccine of Acinetobacter baumannii can reduce the level of inflammation after intraperitoneal lethal dose bacterial challenge; Figure 7 This demonstrates the role of Acinetobacter baumannii recombinant vesicular subunit vaccine in promoting the maturation of bone marrow-derived dendritic cells; Figure 8 The results show that the recombinant vesicular subunit vaccine of Acinetobacter baumannii can effectively induce a bacterial-specific cellular immune response. Figure 9 , Figure 10 , Figure 11 The results show that the Acinetobacter baumannii recombinant vesicular subunit vaccine can effectively induce a strong cytokine response in mouse spleen lymphocytes. Figure 12 The results showed that immunization with the recombinant vesicular subunit vaccine of Acinetobacter baumannii provided some protection against myocardial and hepatic damage when challenged with a lethal dose of the bacteria. Figure 13 The results show that the recombinant vesicular subunit vaccine of Acinetobacter baumannii has good biocompatibility. Detailed Implementation

[0046] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0047] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0048] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0049] Example 1 This embodiment provides a recombinant vesicular subunit vaccine against multidrug-resistant Acinetobacter baumannii. The subunit vaccine uses Acinetobacter baumannii bilayer membrane vesicles (BBV) as a delivery carrier and self-adjuvant. The surface of the Acinetobacter baumannii bilayer membrane vesicles is loaded with three antigenic components: Acinetobacter baumannii outer membrane protein A (OmpA), Acinetobacter baumannii outer membrane protein W (OmpW), and Acinetobacter baumannii outer membrane protein 22 (Omp22).

[0050] The gene sequence of OmpA is shown in SEQ ID NO.1, the gene sequence of OmpW is shown in SEQ ID NO.2, and the gene sequence of Omp22 is shown in SEQ ID NO.3.

[0051] The preparation method of the multidrug-resistant Acinetobacter baumannii recombinant vesicular subunit vaccine is as follows: (1) Preparation of BBV: Clinically isolated multidrug-resistant Acinetobacter baumannii Ab1 strain was cultured in 2000 mL of LB medium at 37°C and 220 rpm for 14 hours. (EDTA-2Na) was slowly added to the culture medium until a concentration of 10 mmol was reached, and then the medium was incubated at 37°C with shaking for 30 minutes. The culture medium was centrifuged at 14,000 rpm for 20 minutes. The supernatant was removed, and the bacterial precipitate was resuspended in 600 mL of HBSS (calcium, magnesium, and phenol red excluding ions). The bacterial suspension was homogenized twice using a high-pressure homogenizer at 1200 Pa, and this process was repeated. The resulting suspension was centrifuged at 6,000 rpm for 30 minutes at 4°C. The supernatant was collected and then centrifuged at 100,000 rpm for 30 minutes at 4°C. The precipitate was resuspended in 2 mL of HBSS and then filtered through a 0.45 μm filter membrane to obtain Acinetobacter baumannii double-membrane vesicle BBV.

[0052] BBV samples prepared from bacteria were first fixed with 2.5% cold glyceraldehyde in 0.2 M calcium phosphate buffer (pH 7.4) for 2 hours, and then fixed with 1% osmium tetroxide in 0.1 M calcium phosphate buffer (pH 7.4) for 1 hour. Five μL of BBV samples were placed on a carbon-coated copper grid, cold-stained with 0.75% (w / v) formic acid, dried for 30 minutes, and then observed and imaged under an 80 kV transmission electron microscope.

[0053] SDS-PAGE and electron microscopy results showed that this embodiment successfully prepared BBV of clinically isolated multidrug-resistant Acinetobacter baumannii (BBV). Figure 1 (D, E, F). Furthermore, after being stored at 4℃ for a certain period of time, the particle size and PDI of this BBV remained very stable. Figure 1 (G).

[0054] (2) Preparation of antigen: The OmpA encoding gene shown in SEQ ID NO.1, the OmpW encoding gene shown in SEQ ID NO.2, and the Omp22 encoding gene shown in SEQ ID NO.3 were inserted into the BamHI (position 5442) and EcoRI (position 5454) restriction sites of the pET-N-His-PreScission-SUMO plasmid, respectively, to construct recombinant plasmids expressing OmpA, OmpW, or Omp22. These plasmids were then transformed into DH5α cells treated with calcium chloride. After induction at 37°C with 1 mmol / L IPTG for 4 hours, the expression of the recombinant proteins was analyzed and identified by 12% heat denaturing SDS-PAGE.

[0055] Each recombinant protein was purified by ultrasonic lysis, nickel ion affinity chromatography, SUMO protease cleavage, and gel filtration desalting to obtain antigens with a purity ≥90%. The purified OmpA, OmpW, and Omp22 antigens were stored at -80℃.

[0056] pET-N-His-PreScission-SUMO is a prokaryotic expression plasmid used to express target proteins with both His and SUMO tags at the N-terminus. The His tag facilitates the purification of the target protein via affinity chromatography, while the SUMO tag helps improve the folding of the target protein and increases its solubility and yield.

[0057] The steps of the nickel ion affinity chromatography are as follows: (a) The chromatography column is equilibrated in buffer 3 and loaded with denatured proteins dissolved in urea; the buffer 3 is 8 M urea, 0.5 M NaCl, and 20 mM urea. pH 8.0.

[0058] (b) Wash the weakly bound proteins from the chromatography column with buffer 4 containing 20 mM imidazole; buffer 4 consists of 0.5 M imidazole, 8 M urea, 0.5 M NaCl, and 20 mM... pH 8.0.

[0059] (c) After returning to buffer 3 to allow the chromatography column to reach equilibrium again, replace pump B of the FPLC with one containing buffer 1 and run at a rate of 1 ml / min using a 20 mL linear gradient from 8 M urea to 0 M urea to change the buffer, while the target outer membrane protein binds to the column. The buffer solution 1 is composed of 5 mM imidazole, 0.5 M NaCl, and 20 mM... pH 8.0.

[0060] (d) After the buffer solution is replaced, change pump A of the FPLC to contain buffer solution 1 and pump B to contain buffer solution 2. Buffer 2 consists of 0.5 M imidazole, 0.5 M NaCl, and 20 mM... pH 7.4.

[0061] (e) The bound proteins were eluted using a gradient of 5–500 mM imidazole to obtain the target outer membrane protein, while His-labeled proteins were eluted with 300–500 mM imidazole.

[0062] OmpA, OmpW, and Omp22 are highly conserved and possess good immunogenicity; the OmpA, OmpW, and Omp22 antigens purified in this example, such as Figure 1 As shown in A, B, and C.

[0063] (3) Loading antigen: The prepared BBV was mixed with three antigens, OmpA, OmpW and Omp22, in a mass ratio of 5μg BBV + 50μg OmpA + 15μg OmpW + 10μg Omp22, and incubated at 4℃-37℃ for 30 minutes to obtain the mixture.

[0064] (4) Ultrasonic treatment: The mixture is ultrasonically treated on ice with an ultrasonic power of 10-30%, each ultrasonic treatment lasts 5-30 minutes, and a total of 2-4 cycles are performed.

[0065] (5) Extrusion purification: The ultrasonic mixture is extruded for 3-10 cycles through an extruder or through a high-pressure homogenizer to load the three antigens OmpA, OmpW and Omp22 onto the Acinetobacter baumannii double membrane vesicles BBV. After purification, the multidrug-resistant Acinetobacter baumannii recombinant vesicle subunit vaccine (VAW22(UE) vaccine) is obtained.

[0066] Electron microscopy results showed that the treated VAW22 (UE) vaccine vesicles had a distinct antigen ring at their edge. Figure 1 H, I). OmpA, OmpW, and Omp22 antigens were successfully presented on BBV ( Figure 1 (J); Compared with BBV alone, VAW22(UE) vaccine has a larger particle size (J). Figure 1 In the middle K and L), the potential increases ( Figure 1 (M).

[0067] Furthermore, in order to investigate whether the antigen was loaded inside or on the surface of the BBV, this embodiment treated the VAW22 (UE) vaccine with proteinase K. The results showed that the protein in the treated sample was completely digested, indicating that the antigen was presented on the surface (N) of the BBV.

[0068] Example 2 This embodiment determined the immunogenicity of the recombinant vesicular subunit vaccine of multidrug-resistant Acinetobacter baumannii described in Example 1 and its cross-recognition characteristics with clinically isolated multidrug-resistant Acinetobacter baumannii, specifically including: To compare the immunogenicity of different vaccine components (using ICR or Balb / c mice, subcutaneously immunized three times, once every week for a total of three times), this embodiment set up different vaccine groups, including PBS group, aluminum adjuvant group, BBV group, OmpA group, OmpW group, Omp22 group, AW22 group (OmpA, OmpW, and Omp22 were physically mixed), VAW22 group (BBV was physically mixed with OmpA, OmpW, and Omp22 without sonication or compression), and VAW22 (UE) group.

[0069] The results showed that one week after the first immunization, the IgM level in the vaccine group containing the antigen increased ( Figure 2 AC (Chinese)

[0070] Following the second and third doses of immunization, antigen-specific antibodies increased in the vaccine-containing groups; among them, the AW22 group, VAW22 group, and VAW22 (UE) group showed better immunogenicity. Figure 2 (In DI). Furthermore, the induced antibodies can recognize other clinically isolated multidrug-resistant Acinetobacter baumannii strains, indicating that the vaccine has potentially broad-spectrum activity. Figure 2 (J).

[0071] Example 3 This embodiment determined the immune bias of the multidrug-resistant Acinetobacter baumannii recombinant vesicular subunit vaccine described in Example 1, specifically including: Enzyme-linked immunosorbent assay (ELISA) was used to analyze the specific IgG1 and IgG2a responses to OmpA, OmpW, and Omp22, respectively. To compare the immune bias induced by different vaccine components, this example measured the IgG1 levels against OmpA, OmpW, and Omp22 in serum after the third immunization in Example 2. Figure 3 (A, B, C) and IgG2a ( Figure 3 The D, E, and F fractions, along with the IgG1 / IgG2a ratio fragment, are used to determine their immune bias. Figure 3 (G, H, I). The results showed that the OmpA-specific immune responses in the OmpA, AW22, VAW22, and VAW22 (UE) groups were Th1-biased. However, the OmpW and Omp22-specific immune responses in different groups were Th2-biased.

[0072] Example 4 This embodiment determined that immunization with the multidrug-resistant Acinetobacter baumannii recombinant vesicular subunit vaccine described in Example 1 could protect mice against clinically isolated lethal doses of multidrug-resistant Acinetobacter baumannii, specifically including: Example 2: Immunized mice were challenged with multidrug-resistant Acinetobacter baumannii to simulate a bacterial sepsis model, thereby evaluating the protection rate of the vaccine.

[0073] The results showed that all vaccine groups provided some degree of protection, but the AW22 and VAW22 (UE) groups provided 100% protection against lethal doses of bacteria. Figure 4 (E, F). We further examined the hearts of mice in different groups ( Figure 4 (G), liver ( Figure 4 (H), spleen ( Figure 4 Middle I), lung ( Figure 4 Middle J), ​​kidney ( Figure 4 (K), blood ( Figure 4 The bacterial load of L) was measured, and the results showed that different vaccine groups could significantly reduce the bacterial load in various organs.

[0074] Both the AW22 group and the VAW22 (UE) group provided 100% protection against lethal doses of bacteria in mice. To further investigate bacterial load in different organs of the two groups, this study analyzed the bacterial load in different organs of the mice in both groups. The results showed that the VAW22 (UE) group significantly reduced bacterial load in the mouse heart (…). Figure 5 (A) Liver ( Figure 5 (B) Spleen ( Figure 5 (C) Lung ( Figure 5 D), kidney ( Figure 5 E), blood ( Figure 5 The bacterial load in F).

[0075] Example 5 This embodiment determined that immunization with the multidrug-resistant Acinetobacter baumannii recombinant vesicular subunit vaccine described in Example 1 could reduce the level of inflammation after intraperitoneal lethal dose bacterial challenge, specifically including: Lethal doses of bacterial infection can induce inflammation in mice. By collecting and testing blood after infection, this example shows that the vaccine group in Example 4, after being challenged with Acinetobacter baumannii, can reduce the level of inflammation in the blood of mice to varying degrees. Figure 6 (A, B, C). Among them, the VAW22(UE) group had the lowest inflammation level ( Figure 6 (D, E, F).

[0076] Example 6 This embodiment measured the effect of the multidrug-resistant Acinetobacter baumannii recombinant vesicular subunit vaccine described in Example 1 on promoting the maturation of bone marrow-derived dendritic cells, specifically including: To compare the effects of different vaccine groups on dendritic cell (DC) maturation in Example 2, bone marrow-derived dendritic cells (BMDCs) were stimulated for 24 hours with phosphate-buffered saline (PBS), AW22, and VAW22 (UE).

[0077] Flow cytometry results showed that the vaccine group significantly increased the expression of co-stimulatory molecules and MHC I, MHC II, and CXCR7. Figure 7 (Acute inflammatory response). Furthermore, treatment with the vaccine also led to an increase in the production of pro-inflammatory cytokines TNF-α and IL-6. Figure 7 The presence of FG in the vaccine group indicates that all vaccine recipients have the potential to activate innate immune responses.

[0078] Example 7 This embodiment determined that the recombinant vesicular subunit vaccine against multidrug-resistant Acinetobacter baumannii described in Example 1 effectively induced a bacterial-specific cellular immune response, specifically including: To evaluate the T-cell immune responses induced by different vaccine groups in Example 2, spleens were harvested and lymphocytes isolated from each 6-8 week old female ICR mouse after immunization according to the above-described procedure and intraperitoneal challenge with a lethal dose. Mouse splenic lymphocytes were stimulated with inactivated clinically isolated multidrug-resistant Acinetobacter baumannii to reflect the level of cellular immunity induced by different vaccines.

[0079] The results showed that the VAW22 (UE) group could induce a significantly increased CD3+ T cell response ( Figure 8 (A, B); different vaccine groups can induce effective CD3+CD8+ T cell responses ( Figure 8 (C, D), and inactivated clinically isolated multidrug-resistant Acinetobacter baumannii can stimulate the proliferation of splenic lymphocytes in different vaccine groups ( Figure 8 (E, F). This embodiment uses , , Stimulation with doses of inactivated bacteria, results showed that The most obvious proliferative effect is achieved by stimulation. Figure 8 (G).

[0080] Example 8 This embodiment determined that the recombinant vesicular subunit vaccine against multidrug-resistant Acinetobacter baumannii described in Example 1 effectively induced a strong cytokine response in mouse splenic lymphocytes, specifically including: This embodiment investigated the protective mechanism of the VAW22 (UE) vaccine using transcriptomics. The results showed that, compared to the AW22 group, the VAW22 (UE) group had more upregulated genes. Figure 9 Genes A and B are mainly associated with epithelial repair, muscle stress and metabolic adaptation, anti-inflammation and anti-oxidation, and complement activation. Downregulated genes are mainly associated with immunosuppression, epithelial repair, coagulation, and metabolic disorders. Figure 9 (C). Analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) also showed that it was mainly related to cytokine-cytokine interactions, cell adhesion molecules, and the IL-17 signaling pathway. Figure 9 D, E and Figure 10 ).

[0081] To further confirm the transcriptome sequencing results, this embodiment used ELISA to analyze splenic lymphocytes after bacterial stimulation to inactivate Th1 cytokines (IFN-γ), Th2 cytokines (IL-4, IL-10), and Th17 cytokines (IL-17A). Figure 11The results showed that, compared with the control group, the vaccine group produced higher levels of cytokine secretion.

[0082] Example 9 This embodiment determined that the recombinant vesicular subunit vaccine against multidrug-resistant Acinetobacter baumannii described in Example 1 provided some protection against myocardial and liver damage after a lethal dose challenge with the bacteria. Specifically, it included: Example 2: Bacterial challenge after vaccine immunization, such as Figure 12 As shown, the levels of AST, CK, and BUN in the vaccine group were lower than those in the control group, suggesting that vaccine immunization has a certain protective effect against liver and myocardial damage.

[0083] Example 10 This embodiment demonstrates that the recombinant vesicular subunit vaccine of multidrug-resistant Acinetobacter baumannii described in Example 1 has good biocompatibility, specifically including: Example 2: Continuous monitoring of body temperature and weight after vaccine immunization, such as... Figure 13 Figures A and B show that the body temperature and weight of mice in the VAW22 (UE) group after immunization were similar to those in the PBS-immunized group, indicating that the vaccine had no significant toxicity. Figure 13 As shown in the CH diagram, there were no significant differences in biochemical indicators. Figure 13 The tissue section results showed that there was no obvious pathological damage in any organ of the VAW22 (UE) vaccine group, indicating that the vaccine has good biocompatibility.

[0084] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A recombinant vesicularized subunit vaccine against multidrug-resistant Acinetobacter baumannii, characterized in that, The subunit vaccine uses Acinetobacter baumannii double membrane vesicles (BBV) as a delivery carrier and self-adjuvant, and the surface load of the Acinetobacter baumannii double membrane vesicles includes at least three antigenic components, including Acinetobacter baumannii outer membrane protein A (OmpA), Acinetobacter baumannii outer membrane protein W (OmpW), and Acinetobacter baumannii outer membrane protein 22 (Omp22).

2. The subunit vaccine according to claim 1, characterized in that, The gene sequence of OmpA is shown in SEQ ID NO.1, the gene sequence of OmpW is shown in SEQ ID NO.2, and the gene sequence of Omp22 is shown in SEQ ID NO.

3.

3. The subunit vaccine according to claim 1, characterized in that, The mass ratio of BBV to OmpA, Omp22, and OmpW is 1-100μg BBV + 1-100μg OmpA + 1-100μg OmpW + 1-100μg Omp22.

4. A method for preparing a recombinant vesicular subunit vaccine of multidrug-resistant Acinetobacter baumannii as described in any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: (1) Preparation of BBV: Multidrug-resistant Acinetobacter baumannii was cultured in a culture medium at 37°C with shaking. EDTA-2Na was added and the culture was continued with shaking. The bacterial precipitate was obtained by centrifugation. The bacterial precipitate was resuspended in an isotonic solution. The bacterial suspension was treated by a high-pressure homogenizer. The resulting suspension was centrifuged at 4°C at a speed of 6000 rpm. The supernatant was collected and centrifuged again at 4°C at a speed of 100000 rpm. The precipitate was resuspended in an isotonic solution and filtered to obtain Acinetobacter baumannii double membrane vesicle BBV. (2) Preparation of antigens: The coding genes of OmpA, OmpW and Omp22 were inserted into prokaryotic expression plasmids to construct recombinant plasmids and transform competent cells. After expression induced by IPTG, OmpA, OmpW and Omp22 antigens with a purity ≥90% were obtained by ultrasonic lysis and nickel ion affinity chromatography. (3) Antigen loading: BBV is mixed with three antigens, OmpA, OmpW and Omp22, according to the set mass ratio and incubated at 4℃-37℃ to obtain a mixture; (4) Ultrasonic treatment: The mixture is ultrasonically treated on ice with an ultrasonic power of 10-30%, each ultrasonic treatment lasts 5-30 minutes, and a total of 2-4 cycles are performed. (5) Extrusion purification: The ultrasonically purified mixture is extruded for 3-10 cycles through an extruder or through a high-pressure homogenizer to load the three antigens OmpA, OmpW, and Omp22 onto the Acinetobacter baumannii double membrane vesicles BBV. After purification, the multidrug-resistant Acinetobacter baumannii recombinant vesicle subunit vaccine is obtained.

5. The use of the recombinant vesicular subunit vaccine of multidrug-resistant Acinetobacter baumannii as described in any one of claims 1 to 3 in the preparation of drugs for the prevention and / or treatment of Acinetobacter baumannii infection.

6. The application according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

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