Acinetobacter baumannii outer membrane protein liposome vaccine and preparation method and application thereof

Abstract

The application discloses a liposome vaccine of Acinetobacter baumannii outer membrane protein and a preparation method and application thereof, and relates to the field of biological medicines. The application efficiently expresses the Acinetobacter baumannii outer membrane protein through a genetic engineering method, and obtains a liposome vaccine of Acinetobacter baumannii outer membrane protein by combining the obtained various outer membrane proteins as antigens with a lipid mixture and an adjuvant. It is verified that the vaccine can provide complete protection for mice attacked by a lethal dose of Acinetobacter baumannii. In the application, the outer membrane protein loaded in the liposome is re-folded in structure, the immunogenicity of the protein is enhanced, and the liposome can reduce the toxicity of the outer membrane protein. Omp38+BauA+FimA can be an effective antigen combination for preventing Acinetobacter baumannii infection. Pam2CSK4 as a TLR2 agonist has a strong auxiliary effect on the liposome vaccine of Acinetobacter baumannii, and can induce the activation of an innate immune response, thereby providing more comprehensive protection for a host.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to an Acinetobacter baumannii outer membrane protein liposome vaccine, its preparation method, and its application. Background Technology

[0002] Acinetobacter baumannii (A. baumannii) is a Gram-negative bacterium that can cause serious infections in critically ill hospital patients, such as ventilator-associated pneumonia, wound infections, and bacteremia, and has become one of the leading pathogens of nosocomial infections. Because Acinetobacter baumannii readily develops resistance to disinfectants and antibiotics, it poses a significant challenge to clinical anti-infective treatment and seriously threatens patients' lives. Vaccines are the most economical and effective means of controlling the spread of pathogens.

[0003] Currently, various forms of Acinetobacter baumannii vaccines are under research, including inactivated whole-cell vaccines, polysaccharide vaccines, protein vaccines, DNA vaccines, subunit vaccines, outer membrane vesicle vaccines, and outer membrane protein complexes (OMCs). All of these vaccine types offer some protection against Acinetobacter baumannii infection in animals, but they are all still in the experimental research stage, and no Acinetobacter baumannii vaccine has yet entered clinical trials.

[0004] The bacterial outer membrane is a double-membrane structure containing a variety of complete membrane proteins. These proteins have many important functions, including nutrient absorption, signal transduction, bacterial adhesion, and waste export. For pathogenic bacteria, outer membrane proteins also play a crucial role in evading host defenses. Therefore, outer membrane proteins are considered the main protective antigens of Acinetobacter baumannii. Outer membrane protein complexes contain abundant antigens, and their use as prophylactic vaccines has been proven to have certain protective and therapeutic effects. However, because they are detached from their "membrane environment," their structure cannot fold correctly, resulting in low immunogenicity and weak immune responses. Furthermore, the excessive variety of antigens in outer membrane protein complexes, the relatively low content of effective antigens, and the excessively high content of endotoxins all limit their vaccine development.

[0005] To obtain single-component outer membrane proteins with high safety, well-defined composition, and high purity, prokaryotic expression vectors were constructed using recombinant DNA technology, and Acinetobacter baumannii outer membrane proteins were induced to express in Escherichia coli. However, most of these proteins still exist in inclusion body form. Due to the complex pathogenic mechanism of Acinetobacter baumannii, with its diverse virulence proteins and pathogenic factors, a single outer membrane protein may only provide limited protection. For example, the recombinant Staphylococcus aureus vaccine uses a multivalent strategy, containing five effective antigens, and is currently in phase III clinical trials. Therefore, multi-component antigens may be an effective strategy for future Acinetobacter baumannii vaccine development.

[0006] The design and development of vaccines generally require adjuvants to assist antigens. Adjuvants can enhance immunogenicity and alter immune response bias, which is crucial for vaccine safety and efficacy. Currently, most Acinetobacter baumannii vaccine development work focuses on outer membrane proteins, but the problem of in vitro misfolding remains unsolved. The screened antigens cannot provide complete protection, and suitable vaccine adjuvants have not yet been found.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to provide an Acinetobacter baumannii outer membrane protein liposome vaccine, its preparation method and application. Experiments have shown that the Acinetobacter baumannii outer membrane protein liposome vaccine can provide complete protection against mice challenged with a lethal dose of Acinetobacter baumannii.

[0009] This invention is implemented as follows:

[0010] In a first aspect, the present invention provides an Acinetobacter baumannii outer membrane protein liposome vaccine comprising liposomes formed from a lipid mixture and Acinetobacter baumannii outer membrane protein loaded onto the lipid mixture, wherein the Acinetobacter baumannii outer membrane protein is selected from at least one of Omp38, BauA, BamA, TamA, FimA and CaW.

[0011] Secondly, the present invention also provides a method for preparing Acinetobacter baumannii outer membrane protein liposome vaccine, which includes: dissolving a mixture of Acinetobacter baumannii outer membrane protein and lipid using a surfactant, and then preparing outer membrane protein liposomes by dialysis.

[0012] Thirdly, the present invention provides the use of the above-described Acinetobacter baumannii outer membrane protein liposome vaccine or the Acinetobacter baumannii outer membrane protein liposome vaccine obtained by the above preparation method in the preparation of products for the prevention and / or treatment of diseases caused by Acinetobacter baumannii.

[0013] The present invention has the following beneficial effects:

[0014] This invention utilizes genetic engineering to efficiently express Acinetobacter baumannii outer membrane proteins. A liposomal vaccine containing these proteins is then developed by combining various outer membrane proteins as antigens with a lipid mixture and adjuvant. The vaccine has been validated to provide complete protection against lethal doses of Acinetobacter baumannii infection in mice. Specifically, the liposomal loading of the outer membrane proteins achieves structural refolding, enhancing protein immunogenicity, and the liposomal loading may reduce the toxicity of the outer membrane proteins. The combination of Omp38, BauA, and FimA may be an effective antigenic combination for preventing Acinetobacter baumannii infection. Pam2CSK4, as a TLR2 agonist, exhibits strong adjuvant activity against the Acinetobacter baumannii liposomal vaccine and may induce activation of the innate immune response, providing more comprehensive protection to the host. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The image shows the nucleic acid electrophoresis results after plasmid digestion in Example 1.

[0017] Figure 2 The results of SDS-PAGE analysis of Omp38, BauA, BamA, TamA, CaW, and FimA on a nickel-passed column with imidazole gradient elution in Example 1 are as follows:

[0018] Figure 3 The results of SDS-PAGE analysis of the five recombinant outer membrane protein liposomes in Example 2 are shown.

[0019] Figure 4 The DLS results for the five recombinant outer membrane protein liposomes in Example 2;

[0020] Figure 5 Transmission electron microscopy images of the five recombinant outer membrane protein liposomes in Example 2;

[0021] Figure 6 The results of the lethal challenge experiment of mice immunized by intramuscular injection of the combined outer membrane protein liposome vaccine in Example 4 are as follows;

[0022] Figure 7 The results of the lethal challenge experiment of mice immunized by intranasal drop of the combined outer membrane protein liposome vaccine in Example 4;

[0023] Figure 8 The SDS-PAGE results of liposomes prepared with different OMC dosages in Experiment Example 1;

[0024] Figure 9 Particle size distribution of liposomes prepared with different OMC dosages in Experiment Example 1;

[0025] Figure 10 The results of SDS-PAGE analysis of Mega-10 and OG dissolved OMC in Experiment Example 2;

[0026] Figure 11 The results of SDS-PAGE analysis of Mega-10 liposomes and OG liposomes in Experiment Example 2 are shown.

[0027] Figure 12 The particle size distribution of liposomes under different ultrasound treatment conditions in Experiment Example 2 is shown.

[0028] Figure 13 SDS-PAGE detection results (Mega-10) of liposomes prepared with different lipid mixture feed ratios in Experiment Example 3;

[0029] Figure 14 The SDS-PAGE (OG) results of liposomes prepared with different lipid mixture feed ratios in Experiment Example 3 are shown.

[0030] Figure 15 The particle size distribution (OG) of liposomes prepared with different lipid mixture feed ratios in Experiment Example 3 is shown.

[0031] Figure 16 This is a particle size distribution diagram of the liposomes in Experiment Example 4 after centrifugation at 1500–5500 rpm;

[0032] Figure 17 The particle size distribution of liposomes in Experiment Example 4 after centrifugation at 5000, 8000, and 13000 rpm is shown below.

[0033] Figure 18 The results of SDS-PAGE analysis of liposomes after centrifugation at 5000, 8000, and 13000 rpm in Experiment Example 4 are shown.

[0034] Figure 19 This is a particle size distribution diagram of liposomes after treatment with carbonate membranes of different pore sizes in Experiment Example 5;

[0035] Figure 20 The results of SDS-PAGE analysis of liposomes treated with carbonate membranes of different pore sizes in Experiment Example 5 are shown.

[0036] Figure 21 The results of SDS-PAGE analysis of liposomes after extrusion and centrifugation in Experiment Example 6;

[0037] Figure 22The particle size distribution of liposomes after extrusion and centrifugation in Experiment Example 6 is shown.

[0038] Figure 23 Transmission electron microscope image of OMC-Lp in Experiment Example 6;

[0039] Figure 24 The serum IgG response level of mice after intramuscular injection of OMC in Experiment Example 7, **P<0.01;

[0040] Figure 25 The survival curves of mice after lethal challenge following intramuscular or intranasal immunization with OMC in Experiment Example 7.

[0041] Figure 26 The values ​​represent the blood and lung bacterial colonization levels in mice immunized with OMC via intramuscular injection in Experiment Example 7 after sublethal challenge. A–B represent the blood and lung bacterial colonization levels in each group of mice. *P<0.05, **P<0.01;

[0042] Figure 27 The values ​​represent the bacterial colonization levels in the blood and lungs of mice immunized with OMC via nasal drops in Experiment Example 7, where A to B represent the bacterial colonization levels in the blood and lungs of mice in each group.

[0043] Figure 28 The IgG response level in the serum of mice immunized with the recombinant protein liposome vaccine in Experiment Example 8 (inactivated whole bacteria coating);

[0044] Figure 29 The survival status of mice immunized with the recombinant protein liposome vaccine in Experiment Example 8 after lethal challenge;

[0045] Figure 30 The serum (×1000) IgG level (inactivated whole bacteria coated) of mice immunized with the combined outer membrane protein liposome vaccine in Experiment Example 8;

[0046] Figure 31 The results of the lethal challenge experiment of mice immunized with the combined outer membrane protein liposome vaccine in Experiment Example 8;

[0047] Figure 32 The serum specific IgG level of mice immunized with the combined outer membrane protein liposome vaccine in Experiment Example 8 after intramuscular injection.

[0048] Figure 33 The serum specific IgG level of mice after intranasal immunization with the combined outer membrane protein liposome vaccine in Experiment Example 8;

[0049] Figure 34 The serum FimA-specific IgG level of mice immunized with the combined outer membrane protein liposome vaccine in Experiment Example 8;

[0050] Figure 35The results of the lethal challenge experiment of mice immunized by intramuscular injection of the combined outer membrane protein liposome vaccine in Experiment Example 8;

[0051] Figure 36 The results of the lethal challenge experiment of mice immunized by intranasal drop of the combined outer membrane protein liposome vaccine in Experiment Example 8 are shown. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0053] Given that current Acinetobacter baumannii vaccines suffer from poorly folded outer membrane proteins, resulting in low immunogenicity, incomplete protection from screened antigens, and the lack of suitable vaccine adjuvants, this invention addresses these shortcomings by designing the following technical solution:

[0054] First, the present invention provides an Acinetobacter baumannii outer membrane protein liposome vaccine, which includes outer membrane protein liposomes and an adjuvant, wherein the outer membrane protein liposomes are obtained by loading Acinetobacter baumannii outer membrane proteins onto a lipid mixture.

[0055] In this invention, the lipid mixture serves to simulate a "membrane environment," loading outer membrane proteins onto a lipid bilayer to improve the problem of incorrect folding of outer membrane protein structures, thereby enhancing immunogenicity. Regarding the composition of the lipid mixture, in some embodiments, the lipid mixture includes phospholipids and cholesterol; the phospholipids may be lecithin, cephalin, soybean phospholipids, and other synthetic phospholipids.

[0056] In some embodiments, the mass ratio of Acinetobacter baumannii outer membrane protein to lipid mixture is 1:1.8-2.2.

[0057] In some embodiments, the mass ratio of cholesterol to lecithin in the lipid mixture is 1:0.8-1.2.

[0058] To further enhance the protective effect of the vaccine, this invention screened for different types of outer membrane protein antigens. The obtained outer membrane protein antigens can be selected from at least one of Omp38, BauA, BamA, TamA, FimA, and CaW. Specifically, the six outer membrane proteins Omp38, BauA, BamA, TamA, FimA, and CaW can be used individually as antigens to prepare the vaccine, or any two, three, four, five, or six of them can be selected in combination as antigens to prepare the vaccine.

[0059] In some embodiments, when the outer membrane protein Omp38 and BauA are combined as an outer membrane protein combination, the resulting vaccine has a better protective effect.

[0060] After further optimization of the technical solution of the present invention, it was found that the vaccine prepared when Omp38, BauA, and FimA are used as an antigen combination has the best protective effect. The dosage ratio of Omp38, BauA, and FimA can be set according to the commonly used antigen dosage for vaccines; the present invention does not limit this. Conventionally, the antigen dosage per mouse is 20 micrograms. In some embodiments, the dosage ratio of antigens Omp38, BauA, and FimA is set to 1:1:1.

[0061] The preparation method for the above outer membrane proteins includes: ligating gene fragments of Omp38, BauA, BamA, TamA, FimA and CaW into expression vectors, respectively, transferring the obtained recombinant vectors into host cells, culturing and inducing them, and then separating and purifying them to obtain recombinant proteins.

[0062] In some embodiments, the expression vector includes pET-30a; the host bacterium includes Escherichia coli BL21(DE3). The expression vector and host bacterium are not limited to the above types, and those skilled in the art can choose according to experimental conditions and actual needs, as long as recombinant protein can be obtained.

[0063] In some embodiments, purification includes performing gel filtration chromatography on the crude product; the packing material in the gel filtration chromatography is Ni-NTA, and the eluent is a urea solution.

[0064] In some embodiments, the adjuvant is selected from at least one of the following: TLR2 agonists such as Pam2CSK4, Pam3CSK4, and FSL-1; TLR4 agonists such as MPLA and GLA; TLR9 agonists such as CpG and Agatolimod; and natural plant adjuvants such as QS-21 and β-glucan. When the antigen is a single outer membrane protein, the adjuvant is preferably MPLA and CpG, more preferably CpG; when the antigen is a combination of multiple outer membrane proteins, the adjuvant is preferably QS-21 and Pam2CSK4; when the antigen is a combination of Omp38, BauA, and FimA outer membrane proteins, the adjuvant is preferably Pam2CSK4.

[0065] In this invention, the ratio of outer membrane protein liposomes to adjuvant can be set according to conventional ratios, and this invention does not limit this.

[0066] The method for preparing Acinetobacter baumannii outer membrane protein liposome vaccine using the above-mentioned mixture of outer membrane proteins, lipids, and adjuvants is as follows:

[0067] The outer membrane protein and lipid mixture of Acinetobacter baumannii were dissolved separately using surfactants, and then the outer membrane protein liposomes were prepared by dialysis. The particle size and particle size distribution can be reduced by means of ultrasound, centrifugation, gradient extrusion and filtration.

[0068] In some embodiments, the surfactant is selected from either decanoyl-N-glucosamide (Mega-10) or octyl-β-D-glucopyranoside (OG); in this invention, when the surfactant is OG, the uniformity of the liposomes and the encapsulation efficiency of the outer membrane proteins can be further improved.

[0069] To improve the uniformity of the prepared liposomes, the amount of outer membrane protein fed in this invention was optimized. Experiments showed that when the feeding ratio was 0.8-1:2, the prepared outer membrane protein liposomes had better uniformity.

[0070] To further improve the encapsulation efficiency of outer membrane proteins, this invention adjusts the amount of lipid mixture added based on the above ratio. Experiments have shown that when the surfactant is OG and the ratio of outer membrane protein to lipid mixture is 1:1-3, the prepared outer membrane protein liposomes have better uniformity and encapsulation efficiency. More preferably, when the ratio of outer membrane protein to lipid mixture is 1:1-1.2, the uniformity and encapsulation efficiency of the outer membrane protein liposomes are even higher.

[0071] To improve the uniformity of liposome particle size, the obtained outer membrane protein liposomes are sonicated after dialysis. In some embodiments, the sonication is probe sonication or water bath sonication. Probe sonication is more vigorous than water bath sonication and may make it easier to obtain liposomes with uniform particle size.

[0072] Centrifugation separates particles of different sizes by utilizing the difference in sedimentation velocity under centrifugal force. In the preparation of outer membrane protein liposomes, by adjusting the centrifugal force, larger or smaller outer membrane protein liposomes can be selectively removed, thereby making the particle size more uniform.

[0073] In some embodiments, the centrifugation speed is 1500-5500 rpm.

[0074] In some embodiments, gradient extrusion is performed by sequentially extruding the outer membrane protein liposomes using 800nm, 400nm, and 200nm carbonate membranes, respectively.

[0075] By optimizing the conditions of centrifugation and gradient extrusion in this invention, more uniform outer membrane protein liposomes with higher encapsulation efficiency can be obtained. The centrifugation conditions are 4000-5500 rpm; the gradient extrusion is performed by sequentially extruding with 800 nm, 400 nm, and 200 nm carbonate membranes respectively.

[0076] The Acinetobacter baumannii outer membrane protein liposome vaccine obtained by the above preparation method can be administered by injection or intranasal drop. Experiments have shown that the vaccine provides complete protection in mice immunized by both intramuscular injection and intranasal drop. Therefore, the vaccine of this invention can be used to prepare products for the prevention and / or treatment of diseases caused by Acinetobacter baumannii, and has good application prospects.

[0077] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0078] Example 1

[0079] This example describes the expression and purification of recombinant proteins Omp38, BauA, BamA, TamA, FimA, and CaW. The specific steps are as follows:

[0080] 1. Construction of recombinant expression plasmids for candidate antigens BauA, BamA, TamA, CaW, and FimA

[0081] The protein sequences of BauA, BamA, TamA, CaW, and FimA were obtained from the UniProt database, derived from Omp38 UniProtKB / TrEMBL:Q6RYW5; BauA UniProtKB / TrEMBL:Q76HJ9; BamA UniProtKB / TrEMBL:A0A7S8WB78; TamA UniProtKB / TrEMBL:D0C629; FimA UniProtKB / TrEMBL:A0A427QVH0; and CaW UniProtKB / TrEMBL:F0KGV8, respectively. Codon optimization was then performed on the sequences of BauA, BamA, TamA, CaW, and FimA (website: http: / / sg.idtdna.com / CodonOpt), and the entire genome was synthesized. Then, the coding sequences were ligated into the pET30a vector using the NdeI and XhoI restriction endonuclease sites, resulting in plasmids pET30a-AbBauA, pET30a-AbBamA, pET30a-AbTamA, pET30a-AbCaW, and pET30a-AbFimA.

[0082] 2. Plasmid transformation

[0083] (1) Melt BL21(DE3) competent cells on ice.

[0084] (2) Take 2 μL of each of the above 4 plasmids and add them to 100 μL of competent cells. Mix them gently and let them stand on ice for 30 min.

[0085] (3) After being heat-shocked in a 42℃ constant temperature metal bath for 90 seconds, it was placed on ice for 3 minutes.

[0086] (4) Add 500 μL of antibiotic-free LB medium to each tube and incubate at 37°C and 220 rpm for 1 h with shaking.

[0087] (5) Centrifuge at 3000 rpm for 10 min at 4℃, and resuspend the precipitate in 100-200 μL of LB medium.

[0088] (6) Spread the bacterial solution on LB plates with 50 μg / mL kanamycin resistance and incubate overnight at 37°C to grow single colonies.

[0089] 3. Recombinant protein-induced expression

[0090] (1) Pick a single colony from an LB plate with a 10 μL pipette tip and inoculate it into 30 mL of LB liquid medium (50 μg / mL kanamycin resistant) and incubate overnight at 37°C and 220 rpm.

[0091] (2) Expand the bacterial culture to 3 L LB liquid medium (40 μg / mL kanamycin resistance) at a ratio of 1:100. Continue culturing at 37℃ and 220 rpm for 3 h until the bacterial culture reaches OD. 600nm It is 0.8.

[0092] (3) Add 3 mL of 1 M IPTG inducer and induce culture at 37 °C and 220 rpm for 4 h.

[0093] (4) Centrifuge at 5000 rpm for 20 min and collect the bacterial precipitate.

[0094] (5) Add PBS at a ratio of 1:10 (w / v) to resuspend the bacterial cells.

[0095] (6) Sterilization: Sterilize in a homogenizer at 750 bar for 10 min.

[0096] (7) Centrifuge at 4℃ and 5000rpm for 10min and take the supernatant. Centrifuge at 4℃ and 12000rpm for 30min and take the precipitate, which is the inclusion body.

[0097] 4. Recombinant protein purification and concentration

[0098] (1) Dissolve the precipitate with 10-20 mL of 8M urea.

[0099] (2) The Ni-NTA-filled affinity chromatography column was washed three times with 20 mL of PBS, and then equilibrated three times with 20 mL of 8 M urea.

[0100] (3) Add the inclusion body solution to the affinity chromatography column and let it stand for about 30 minutes.

[0101] (4) Elution: After eluting the inclusion bodies, collect them and then elute the nickel column with 15 mL each of 25 mM, 40 mM, 60 mM, 80 mM, 100 mM, 200 mM, 300 mM, 500 mM and 1 M imidazole solutions, and collect the eluent.

[0102] (5) Take 10 μL of the eluent from each step and perform SDS-PAGE.

[0103] (6) Select an ultrafiltration tube with a suitable pore size, add pure water to submerge the filter membrane, place it on ice to pre-cool for 2-5 minutes, and then pour out the pure water.

[0104] (7) Pour the eluent with high purity of the target band into the ultrafiltration tube in sequence, and centrifuge at 3000 rpm until concentrated to the required volume.

[0105] (8) Protein concentration was determined by the BCA method.

[0106] The plasmid was identified by double digestion with Nde I and Xho I. The electrophoresis results of the nucleic acid after plasmid digestion are as follows: Figure 1 As shown, the molecular weight of the fragment is consistent with expectations. Figure 1 In the assay, lane M (5 μL / well): DNA marker; lane 1 (5 μL / well): pET30a-AbBauA; lane 2 (5 μL / well): pET30a-AbFimA; lane 3 (5 μL / well): pET30a-AbCaW; lane 4 (5 μL / well): pET30a-AbBamA; lane 5 (5 μL / well): pET30a-AbTamA.

[0107] Plasmids pET30a-AbBauA, pET30a-AbBamA, pET30a-AbTamA, pET30a-AbCaW, and pET30a-AbFimA, which were correctly identified by enzyme digestion, were transformed into BL21(DE3) competent cells. Induction conditions of 1 mM IPTG, 37℃, and 220 rpm were used, and all proteins were expressed in inclusion body form. Based on previous work, recombinant protein Omp38 was directly induced from preserved glycerol bacteria. The molecular weights of the proteins were calculated based on the nucleic acid sequence lengths: Omp38 was approximately 38 kDa, BauA was approximately 79 kDa, BamA was approximately 92 kDa, TamA was approximately 96 kDa, CaW was approximately 52 kDa, and FimA was approximately 19 kDa. The recombinant proteins were purified using Ni-NTA affinity chromatography, and the eluent was subjected to SDS-PAGE. The results are as follows: Figure 2 As shown, the molecular weight is consistent with expectations.

[0108] Figure 2The AF values ​​correspond to the results of SDS-PAGE analysis of Omp38, BauA, BamA, TamA, CaW, and FimA on nickel-coated columns with imidazole gradient elution. In 2A, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): bacterial culture after IPTG induction; lane 2 (10 μL / well): centrifuged precipitate after lysis; lane 3 (10 μL / well): supernatant from centrifuged inclusion bodies; lane 4 (10 μL / well): protein... White flow-through buffer; Lane 5 (10 μL / well): 25 mM imidazole elution buffer; Lane 6 (10 μL / well): 40 mM imidazole elution buffer; Lane 7 (10 μL / well): 60 mM imidazole elution buffer; Lane 8 (10 μL / well): 80 mM imidazole elution buffer; Lane 9 (10 μL / well): 100 mM imidazole elution buffer; Lane 10 (10 μL / well): 200 mM imidazole elution buffer; Lane 11 (10 μL / well): 300 mM imidazole elution buffer.

[0109] In lane 2B, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): bacterial culture before IPTG induction; lane 2 (10 μL / well): bacterial culture after IPTG induction; lane 3 (10 μL / well): supernatant from centrifuged inclusion bodies; lane 4 (10 μL / well): inclusion bodies; lane 5 (10 μL / well): protein flow-through medium; lane 6 (10 μL / well): 25 mM imidazole. Lane 7 (10 μL / well): 40 mM imidazole elution buffer; Lane 8 (10 μL / well): 60 mM imidazole elution buffer; Lane 9 (10 μL / well): 80 mM imidazole elution buffer; Lane 10 (10 μL / well): 100 mM imidazole elution buffer; Lane 11 (10 μL / well): 200 mM imidazole elution buffer; Lane 12 (10 μL / well): 300 mM imidazole elution buffer.

[0110] In lane 2C, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): protein flow-through buffer; lane 2 (10 μL / well): 25 mM imidazole elution buffer; lane 3 (10 μL / well): 40 mM imidazole elution buffer; lane 4 (10 μL / well): 60 mM imidazole elution buffer; lane 5 (10 μL / well): 80 mM imidazole elution buffer; lane 6 (10 μL / well): 100 mM imidazole elution buffer; lane 7 (10 μL / well): 200 mM imidazole elution buffer; lane 8 (10 μL / well): 300 mM imidazole elution buffer.

[0111] In 2D, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): protein flow-through buffer; lane 2 (10 μL / well): 25 mM imidazole elution buffer; lane 3 (10 μL / well): 40 mM imidazole elution buffer; lane 4 (10 μL / well): 60 mM imidazole elution buffer; lane 5 (10 μL / well): 80 mM imidazole elution buffer; lane 6 (10 μL / well): 100 mM imidazole elution buffer; lane 7 (10 μL / well): 200 mM imidazole elution buffer.

[0112] In lane 2E, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): lysed supernatant; lane 2 (10 μL / well): inclusion body supernatant; lane 3 (10 μL / well): protein flow-through buffer; lane 4 (10 μL / well): 25 mM imidazole elution buffer; lane 5 (10 μL / well): 40 mM imidazole elution buffer; lane 6 (10 μL / well): 60 mM imidazole elution buffer. mM imidazole elution buffer; Lane 7 (10 μL / well): 80 mM imidazole elution buffer; Lane 8 (10 μL / well): 100 mM imidazole elution buffer; Lane 9 (10 μL / well): 200 mM imidazole elution buffer; Lane 10 (10 μL / well): 300 mM imidazole elution buffer; Lane 11 (10 μL / well): 500 mM imidazole elution buffer; Lane 12 (10 μL / well): 1 M imidazole elution buffer.

[0113] Lane 2F is as follows: Lane M (5 μL / well): pre-stained protein standard molecular weight; Lane 1 (10 μL / well): 25 mM imidazole elution buffer; Lane 2 (10 μL / well): 40 mM imidazole elution buffer; Lane 3 (10 μL / well): 60 mM imidazole elution buffer; Lane 4 (10 μL / well): 80 mM imidazole elution buffer; Lane 5 (10 μL / well): 100 mM imidazole elution buffer; Lane 6 (10 μL / well): 200 mM imidazole elution buffer; Lane 7 (10 μL / well): 300 mM imidazole elution buffer; Lane 8 (10 μL / well): 500 mM imidazole elution buffer; Lane 9 (10 μL / well): 1 M imidazole elution buffer.

[0114] Example 2

[0115] This embodiment describes the preparation and characterization of recombinant outer membrane protein liposomes.

[0116] The preparation methods for each outer membrane protein liposome are as follows:

[0117] (1) Add 1 mg of outer membrane protein to 20% OG to make its concentration 1%. Mix outer membrane protein, cholesterol and lecithin (dissolved in chloroform) in a mass ratio of 1:1:1 and incubate at room temperature for 30 min.

[0118] (2) At 4℃, dialyze the mixed solution against PBS for 24 hours, changing the solution twice during the process.

[0119] (3) Ice bath, 150w, probe ultrasound liposome 10min (run for 15s, pause for 5s).

[0120] (4) The liposomes were treated with 800nm, 400nm and 200nm carbonate membranes in sequence, squeezed 21 times and then removed from the syringe on the other side.

[0121] (5) Centrifuge the liposomes at 4000 rpm for 10 min and collect the supernatant.

[0122] (6) Use SDS-PAGE to view the stripes.

[0123] SDS-PAGE results are as follows Figure 3 As shown, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): Omp38-Lp; lane 2 (10 μL / well): BauA-Lp; lane 3 (10 μL / well): BamA-Lp; lane 4 (10 μL / well): TamA-Lp; lane 5 (10 μL / well): FimA-Lp. Each protein band is clearly visible. DLS results and protein encapsulation efficiency are shown below. Figure 4 As shown in Table 1, the particle size of each recombinant protein liposome is around 200 nm, and the PDI is less than 0.3. Figure 5 Transmission electron microscopy images of five recombinant proteins: A: Omp38-Lp; B: BauA-Lp; C: BamA-Lp; D: TamA-Lp; E: FimA-Lp. The liposomes are all regular spherical vesicles, which meet the requirements for animal immunization experiments.

[0124] Table 1 Characterization of five outer membrane protein liposomes

[0125]

[0126]

[0127] Example 3

[0128] This embodiment describes a method for preparing an Acinetobacter baumannii outer membrane protein liposome vaccine. The specific steps are as follows:

[0129] (1) The outer membrane protein and lipid mixture were dissolved separately using a surfactant. The outer membrane protein was a combination of Omp38, BauA and FimA with a mass ratio of 1:1:1 obtained in Example 1. The mass ratio of the outer membrane protein to the lipid mixture was 1:1. The lipid mixture was composed of lecithin and cholesterol with a mass ratio of 1:1. The surfactant was OG.

[0130] (2) Transfer the solution of outer membrane protein and lipid mixture to a dialysis bag with a molecular weight cutoff of 8-14KD and dialyze for 24-36 hours;

[0131] (3) After dialysis, the product was subjected to sonication, centrifugation and gradient extrusion. The sonication power was 150w for 10min and the centrifugation speed was 4000rpm. The product was extruded sequentially using 800nm, 400nm and 200nm carbonate membranes respectively. The amount of vaccine used per mouse Pam2CSK4 was 7.5 micrograms and the amount of antigen used per mouse was 20 micrograms.

[0132] Example 4

[0133] This example evaluates the protective efficacy of the Acinetobacter baumannii outer membrane protein liposome vaccine, as detailed below:

[0134] Mice were immunized by intramuscular injection and intranasal administration of Omp38-Lp, BauA-Lp, and FimA-Lp obtained in Example 3, combined with Pam2CSK4 adjuvant. Fourteen days after the last immunization, mice were challenged with a lethal dose of LAC-4. The results of the lethal dose challenge are as follows: Figure 6-7 As shown, the vaccine prepared in Example 3 achieved complete protection in mice immunized by both intramuscular injection and intranasal administration.

[0135] Experimental Example 1

[0136] This experiment compared the performance of liposomes obtained from Acinetobacter baumannii outer membrane protein complex (OMC, obtained by extraction) at different feed amounts. The other steps in the liposome preparation method were the same as in Example 3. Three experimental groups were set with OMC to lipid mixture mass ratios of 1 mg:2 mg, 0.5 mg:2 mg, and 0.25 mg:2 mg. The SDS-PAGE results of the prepared liposomes are shown below. Figure 8 As shown, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): 1 mg OMC-Lp (unfiltered); lane 2 (10 μL / well): 1 mg OMC-Lp (filtered); lane 3 (10 μL / well): 0.5 mg OMC-Lp (unfiltered); lane 4 (10 μL / well): 0.5 mg OMC-Lp (filtered); lane 5 (10 μL / well): 0.25 mg OMC-Lp (unfiltered); lane 6 (10 μL / well): 0.25 mg OMC-Lp (filtered).

[0137] Before filtration, the protein bands in the liposomes prepared at each feed rate were clear, and the overall content was proportional to the feed rate. However, after filtration, no protein was observed in the liposomes at any feed rate, suggesting a high proportion of free OMC and large-particle OMC-Lp in the system.

[0138] PDI is an important reference index for describing the uniformity of particle distribution. The larger the value, the more non-uniform the particle size distribution. A PDI < 0.3 is considered to be a system with relatively uniform particle size. Figure 9 The DLS assay results showed that before filtration, OMC-Lp mostly had a single peak, and the particle distribution was disordered and uneven. Filtration can improve the liposome particle size and PDI. The particle size and PDI of 1 mg OMC-Lp were better than those of 0.25 mg OMC-Lp and 0.5 mg OMC-Lp (Table 1).

[0139] Table 2 Characterization of liposomes prepared with different OMC dosages

[0140]

[0141] Based on the above results, liposomes were prepared by dialysis of a protein feed of 1 mg and a lipid mixture of 2 mg.

[0142] Experimental Example 2

[0143] This experimental example is a screening experiment for surfactants and a comparison of ultrasonic methods. The other steps in the preparation method of liposomes are the same as in Example 2.

[0144] Mega-10 and OG, two commonly used surfactants for dissolving outer membrane proteins, were selected to improve the homogeneity of liposomes and the encapsulation efficiency of outer membrane proteins. Liposomes were prepared using Mega-10 and OG respectively. First, their solubilizing effects on OMC were compared. OMC was dissolved using Mega-10 and OG respectively, and the supernatant was subjected to SDS-PAGE after centrifugation. The results are as follows: Figure 10 As shown, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): 1 mg / mL OMC; lane 2 (10 μL / well): Mega-10 OMC; lane 3 (10 μL / well): OG OMC.

[0145] Figure 10 The results showed no significant difference in the solubility of OMC between the two surfactants. Liposomes were then prepared by dialysis, with the protein-to-lipid mixture ratio as described above.

[0146] To improve the uniformity of liposome particle size, the liposomes were sonicated in a 37°C water bath for 1 hour after dialysis, followed by filtration through a filter membrane. SDS-PAGE results are shown below. Figure 7 As shown, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): 1 mg / mL OMC; lane 2 (10 μL / well): Mega-10 OMC-Lp; lane 3 (10 μL / well): OG OMC-Lp.

[0147] Figure 11 SDS-PAGE results showed that the protein bands of Mega-10 liposomes and OG liposomes were not clear, indicating that the protein content in the liposomes was low and the protein encapsulation efficiency of the liposomes needs to be further improved. Figure 12 The DLS results showed that OGBlank-Lp and OMC-Lp had better particle size and PDI than Mega-10 Blank-Lp and OMC-Lp, indicating that water bath ultrasound can improve the uniformity of liposomes to a certain extent, but the effect on Mega-10 liposomes is not good.

[0148] Probe ultrasound is more intense than water bath ultrasound, potentially making it easier to obtain liposomes with uniform particle size. Under otherwise identical conditions, probe ultrasound was used instead of water bath ultrasound to process liposomes. Compared to water bath ultrasound, probe ultrasound improved the uniformity of liposomes; the particle size of Mega-10 OMC-Lp obtained by water bath ultrasound was 563.7±274.0 nm, with a PDI of 0.768±0.241; while the particle size obtained by probe ultrasound was 177.2±17.7 nm, with a PDI of 0.461±0.095. The particle size of OG OMC-Lp obtained by water bath ultrasound was 247.4±9.2 nm, with a PDI of 0.322±0.026; while the particle size obtained by probe ultrasound was 162.1±9.5 nm, with a PDI of 0.305±0.040 (Table 3).

[0149] Table 3 Characterization of liposomes under different ultrasound treatment conditions

[0150]

[0151]

[0152] The liposomes prepared based on OG had better particle size and PDI than Mega-10, indicating that OG has a better effect on improving liposome particle size, and probe ultrasound can effectively improve the uniformity of liposomes.

[0153] Experimental Example 3

[0154] This experiment compares the performance of liposomes obtained from lipid mixtures at different feed amounts. 1, 2, and 3 times the amount of lipid mixture were mixed with OMC, then dissolved in Mega-10 and OG, respectively. Liposomes were prepared by dialysis and further enhanced by treatment with a liposome extruder (200 nm carbonate membrane).

[0155] Liposomes were prepared using the Mega-10 dialysis method. The encapsulation efficiencies of liposomes prepared from 1-, 2-, and 3-fold lipid mixtures, as determined by the BCA method, were 33.5%, 18.3%, and 33.0%, respectively. SDS-PAGE results are shown below. Figure 13As shown, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): 1-fold lipid mixture Mega10 OMC-Lp; lane 2 (10 μL / well): 1-fold lipid mixture Mega10 OMC-Lp (filtered); lane 3 (10 μL / well): 2-fold lipid mixture Mega10 OMC-Lp; lane 4 (10 μL / well): 2-fold lipid mixture Mega10 OMC-Lp (filtered); lane 5 (10 μL / well): 3-fold lipid mixture Mega10OMC-Lp; lane 6 (10 μL / well): 3-fold lipid mixture Mega10 OMC-Lp (filtered).

[0156] Figure 13 SDS-PAGE results showed that after OMC was prepared into liposomes, the main protein bands changed, especially the protective antigen Omp38 protein disappeared. Therefore, the use of Mega-10 to prepare liposomes was discontinued.

[0157] Liposomes were prepared using the OG dialysis method. The encapsulation efficiencies of liposomes prepared from 1, 2, and 3-fold lipid mixtures, as determined by the BCA method, were 55.7%, 55.7%, and 40.5%, respectively. SDS-PAGE results are shown below. Figure 14 As shown, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): 1-fold lipid mixture OG OMC-Lp; lane 2 (10 μL / well): 1-fold lipid mixture OG OMC-Lp (extruder treatment); lane 3 (10 μL / well): 1-fold lipid mixture OG OMC-Lp (filtered); lane 4 (10 μL / well): 2-fold lipid mixture OG OMC-Lp; lane 5 (10 μL / well): 2-fold lipid mixture OG OMC-Lp (filtered); lane 6 (10 μL / well): 3-fold lipid mixture OG OMC-Lp; lane 7 (10 μL / well): 3-fold lipid mixture OG OMC-Lp (extruder treatment); lane 8 (10 μL / well): 3-fold lipid mixture OG OMC-Lp (filtered).

[0158] Figure 14 SDS-PAGE results showed that the main band was prominent and each band was clear before extruder treatment. Bands could also be observed after extruder treatment and filtration. The concentration of the main protein band was similar in the 1-fold and 2-fold lipid mixtures, but higher than that in the 3-fold lipid mixture group.

[0159] Figure 15The DLS measurements showed that the OMC-Lp particles prepared with a 1-fold lipid mixture had a particle size of 505.7±10.5 nm and a PDI of 0.320±0.041; the OMC-Lp particles prepared with a 2-fold lipid mixture had a particle size of 391.5±12.2 nm and a PDI of 0.532±0.079; and the OMC-Lp particles prepared with a 3-fold lipid mixture had a particle size of 356.7±10.2 nm and a PDI of 0.433±0.022 (Table 3).

[0160] Table 4. Characterization of liposomes prepared with different lipid mixture feed ratios (OG)

[0161]

[0162] The above results indicate that the liposomes prepared from the 1-fold lipid mixture have the lowest PDI, and the 1-fold liposome mixture will be used in subsequent experiments.

[0163] Experiment Example 4

[0164] This experiment demonstrates how centrifugation improves the particle size distribution of liposomes. Differential centrifugation separates particles of different sizes by utilizing the difference in sedimentation velocity under centrifugal force. During liposome preparation, adjusting the centrifugal force allows for the selective removal of larger or smaller liposomes, resulting in liposomes with more uniform particle size.

[0165] This experiment used differential centrifugation to process liposomes to investigate whether it could improve the homogeneity of OMC liposomes. Liposomes were obtained after dialysis and sonication according to the preparation method in Example 2, and centrifuged at 1500 rpm, 2500 rpm, 3500 rpm, 4500 rpm, and 5500 rpm for 5 min, respectively. The particle size distribution was measured, and the results are as follows: Figure 16 As shown.

[0166] The characterization data of liposomes before and after centrifugation are shown in Table 5:

[0167] Table 5 Characterization of liposomes after centrifugation at 1500–5500 rpm

[0168]

[0169] After centrifugation at 5500 rpm, the particle size of liposomes decreased from 1148.9±277.1 nm to 294.2±15.2 nm, and the PDI decreased from 0.993±0.013 to 0.413±0.057, indicating that differential centrifugation has a certain effect on improving the uniformity of liposomes.

[0170] Further increasing the centrifugation speed to 5000 rpm, 8000 rpm, and 13000 rpm resulted in liposome particle sizes all below 200 nm and PDI all below 0.3. Figure 17 (Table 6), but the SDS-PAGE results showed that the protein bands at each rotation speed were no longer clear, indicating that OMC loss was severe at high rotation speeds. Figure 18 ). Figure 13 Lane M (5 μL / well): Pre-stained protein standard molecular weight; Lane 1 (10 μL / well): 5000 rpm OMC-Lp; Lane 2 (10 μL / well): 8000 rpm OMC-Lp; Lane 3 (10 μL / well): 13000 rpm OMC-Lp.

[0171] Table 6 Characterization of liposomes after centrifugation at 5000, 8000, and 13000 rpm

[0172]

[0173] Experimental Example 5

[0174] This experiment aimed to improve the particle size distribution of liposomes through gradient extrusion. While high-speed centrifugation helps improve the particle size distribution, protein loss also increases with increasing rotational speed. Previous experiments have demonstrated that extruders have a certain effect on particle size improvement; therefore, this experiment attempted to treat liposomes sequentially with 800 nm, 400 nm, and 200 nm carbonate membranes.

[0175] Figure 19 The results in Table 7 show that the particle size and PDI of liposomes both decreased with decreasing carbonate membrane pore size. However, even when liposomes were extruded using a 200 nm carbonate membrane, the particle size still reached 494.7 nm. Obvious bands were still observed after filtration through a 0.45 μm membrane, with a protein encapsulation efficiency of 45.04%. The bands disappeared after filtration through a 0.22 μm membrane, indicating significant protein loss. Figure 20 ). Figure 20 Lane M (5 μL / well): Pre-stained protein standard molecular weight; Lane 1 (10 μL / well): 800 nm carbonate membrane OMC-Lp; Lane 2 (10 μL / well): 400 nm carbonate membrane OMC-Lp; Lane 3 (10 μL / well): 200 nm carbonate membrane OMC-Lp; Lane 4 (10 μL / well): 0.45 μm OMC-Lp; Lane 5 (10 μL / well): 0.22 μm OMC-Lp.

[0176] Table 7 Characterization of liposomes after treatment with carbonate membranes of different pore sizes.

[0177]

[0178] Experimental Example 6

[0179] This experiment aimed to improve liposome particle size distribution through a combination of gradient extrusion and differential centrifugation. To obtain liposomes with uniform particle size and high protein loading, the combination of gradient extrusion and differential centrifugation was explored. Liposomes were prepared using the dialysis method described in Example 2 and then sequentially treated with 800 nm, 400 nm, and 200 nm carbonate membranes. SDS-PAGE results showed intact protein bands. Figure 21 ). Figure 20 Lane M (5 μL / well): Pre-stained protein standard molecular weight; Lane 1 (10 μL / well): OMC-Lp; Lane 2 (10 μL / well): 800 nm OMC-Lp; Lane 3 (10 μL / well): 400 nm OMC-Lp; Lane 4 (10 μL / well): 200 nm OMC-Lp.

[0180] The DLS results are as follows: Figure 22 As shown in Table 7, after centrifugation at 2000 rpm, the particle size of OMC-Lp was 202.5 ± 6.1 nm, the PDI was 0.308 ± 0.045, and the encapsulation efficiency was 71.6%; after centrifugation at 4000 rpm, the particle size of OMC-Lp was 188.3 ± 4.1 nm, the PDI was 0.278 ± 0.048, and the protein encapsulation efficiency was 52.2%. Therefore, a centrifugation speed of 4000 rpm was used as one of the conditions for subsequent liposome treatment.

[0181] Table 8 Characterization of liposomes after extrusion and centrifugation.

[0182]

[0183] Its morphology and structure were observed using transmission electron microscopy. Figure 23 It appears as regular vesicles, meeting the requirements for animal immunization experiments.

[0184] Experimental Example 7

[0185] This experiment evaluates the protective effect of OMC liposomes against Ab infection.

[0186] The OMC-Lp vaccine was constructed by combining it with MPLA and CpG adjuvants, and its protective effect against lethal doses of Acinetobacter baumannii challenge was evaluated after immunization.

[0187] Mice were immunized with Ab OMC liposome vaccine via intramuscular injection and intranasal administration. OMC liposomes containing 20 μg of protein, combined with MPLA or CpG adjuvant, were administered via intramuscular injection or intranasal administration three times at days 0, 14, and 28. Ab challenge was performed 14 days after the last immunization to evaluate the vaccine's protective effect against Ab infection.

[0188] (1) Serum IgG response

[0189] ELISA was used to detect the level of OMC-specific IgG in the serum of mice immunized by intramuscular injection, such as... Figure 24 As shown, the OMC group, OMC-Lp group and OMC-Lp-MPLA group all produced significant specific IgG antibody responses, but there was no significant difference in IgG levels among the groups, indicating that liposome loading and the addition of adjuvants failed to significantly increase serum IgG levels.

[0190] (2) Evaluation of the effectiveness of protection against viral attack

[0191] Mice in the intramuscular injection immunization group were challenged with a sublethal dose 14 days after the last immunization. Intramuscular injection of OMC-Lp+CpG only provided a 37.5% survival rate. Figure 25 Results of bacterial colonization experiment () Figure 26 The results showed that the blood bacterial load in the OMC-Lp group and the OMC-Lp+MPLA group was significantly lower than that in the PBS group. No bacteria were found in the lungs of the OMC-Lp group.

[0192] Mice in the intranasal immunization group were challenged with a sublethal dose of the virus 14 days after the last immunization. Results of the bacterial colonization experiment (...) Figure 27 The results showed that the bacterial colonization in the blood of the OMC-Lp+MPLA group showed a decreasing trend, and there was no statistically significant difference in the bacterial colonization in the blood and lungs among the groups.

[0193] Experimental Example 8

[0194] Previous studies have shown that single outer membrane protein liposomes are more suitable for combination with CpG adjuvants, while combined outer membrane protein liposomes are more suitable for combination with QS-21 adjuvants and Pam2CSK4 adjuvants. Therefore, in this experiment, when screening adjuvants, we directly combined single outer membrane protein liposomes with CpG adjuvants, and combined outer membrane protein liposomes with QS-21 adjuvants and Pam2CSK4 adjuvants, to investigate their protective effects against lethal doses of Acinetobacter baumannii challenge.

[0195] 1. Evaluation of the protective efficacy of single outer membrane protein liposome vaccines

[0196] 20 μg of protein-quantified Omp38-Lp, BauA-Lp, BamA-Lp, and TamA-Lp were mixed with 20 μg of CpG and administered intramuscularly to mice at 0, 14, and 28 days. Mice were challenged with a lethal dose of the virus at 42 days. Blood was collected from the tail vein of mice before challenge to measure serum antibody titers.

[0197] (1) Serum IgG response

[0198] The 96-well plates were coated with inactivated LAC-4 whole bacteria, and the ELISA results were as follows. Figure 28As shown, A: IgG response level of mouse serum diluted 100-fold; B: IgG response level of mouse serum diluted 1000-fold; C: IgG response level of mouse serum diluted 4000-fold. At a dilution factor of ×100, the IgG levels in the recombinant protein group were significantly higher than those in the PBS group (P<0.01, P<0.001, or P<0.0001). The BauA-Lp+CpG and BamA-Lp+CpG groups maintained high IgG levels at dilution factors of ×1000 and ×4000, showing significant differences compared to the PBS group (P<0.05 or P<0.01).

[0199] (2) Evaluation of protection effect

[0200] like Figure 29 As shown in Figure A, 14 days after the last immunization, mice were challenged with a lethal dose of LAC-4. The Blank-Lp+CpG group recovered the fastest body weight, followed by the BauA-Lp+CpG group and the Omp38-Lp+CpG group. Due to missing records of deaths in the TamA-Lp+CpG group after challenge, the BamA-Lp+CpG group experienced the largest decrease in body weight and the slowest recovery. Survival outcomes after challenge (…) Figure 29 B) shows that the survival rate of each group was poor, with a survival rate of 20% in the Omp38-Lp+CpG group and 10% in the Blank-Lp+CpG and BamA-Lp+CpG groups.

[0201] 2. Evaluation of the protective efficacy of Omp38-Lp, BauA-Lp, BamA-Lp, and TamA-Lp recombinant protein liposome vaccines

[0202] like Figure 29 As shown in Figure A, Omp38-Lp and BauA-Lp alone resulted in minimal changes in mouse body weight after immunization-induced challenge. Analysis of mouse survival revealed a 20% survival rate in the Omp38-Lp group, with the BauA-Lp, BamA-Lp, and TamA-Lp groups showing progressively faster mortality rates. Therefore, the recombinant protein combinations were determined to be: Omp38-Lp group, Omp38-Lp+BauA-Lp group, Omp38-Lp+BauA-Lp+BamA-Lp group, and Omp38-Lp+BauA-Lp+BamA-Lp+TamA-Lp group.

[0203] The combined outer membrane protein liposomes (each protein content 20 μg) were used to immunize mice at 0, 14, and 28 days. After the three immunizations, tail vein blood was collected from the mice to measure serum IgG levels.

[0204] (1) Serum IgG response

[0205] The IgG level was detected by coating 96-well plates with inactivated whole bacteria. Figure 30 As shown, all experimental groups showed statistically significant differences compared to the PBS group. With the increase in the types of recombinant proteins used in immunization, IgG levels tended to rise, with the highest IgG levels observed in the Omp38-Lp+BauA-Lp+BamA-Lp+TamA-Lp group.

[0206] (2) Evaluation of protection effect

[0207] Mice were immunized intramuscularly with Omp38-Lp, BauA-Lp, BamA-Lp, and TamA-Lp combined with QS-21 adjuvant. Fourteen days after the last immunization, mice were challenged with a lethal dose of LAC-4. The results of the lethal dose challenge showed ( Figure 31 The PBS group and the Omp38-Lp+BauA-Lp+BamA-Lp+TamA-Lp group died first, while the Omp38-Lp group died the slowest. The mice in all groups showed poor resistance to LAC-4 infection. Based on the analysis of the mice's mortality rate, Omp38 and BauA are considered as potentially effective antigens for further research.

[0208] ELISA analysis showed that the levels of Omp38 and BauA-specific antibodies in the three-component liposome vaccine were higher than those in the two-component liposome vaccine. Figure 32 , Figure 33 The levels of IgG antibodies in the intramuscular injection group were higher than those in the nasal drop immunization group. Figure 34 ). Figure 35 and 36 This indicates that the Omp38-Lp+BauA-Lp+FimA-Lp group can completely protect mice from lethal doses of LAC-4 challenge when administered via intramuscular injection or intranasal immunization.

[0209] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A Acinetobacter baumannii outer membrane protein liposome vaccine, characterized in that, It includes liposomes and adjuvants, wherein the liposomes are formed from a lipid mixture and Acinetobacter baumannii outer membrane proteins loaded onto the lipid mixture; The outer membrane protein in the Acinetobacter baumannii outer membrane protein liposome vaccine is a combination of Omp38 and BauA, and its adjuvant is QS-21 or Pam2CSK4; or The outer membrane protein in the Acinetobacter baumannii outer membrane protein liposome vaccine is a combination of Omp38, BauA and FimA, and its adjuvant is Pam2CSK4.

2. The Acinetobacter baumannii outer membrane protein liposome vaccine according to claim 1, characterized in that, The mass ratio of the Acinetobacter baumannii outer membrane protein to lipid mixture is 1:1.8-2.

2.

3. The Acinetobacter baumannii outer membrane protein liposome vaccine according to claim 1, characterized in that, The lipid mixture contains cholesterol and lecithin.

4. The Acinetobacter baumannii outer membrane protein liposome vaccine according to claim 1, characterized in that, The mass ratio of cholesterol to lecithin in the lipid mixture is 1:0.8-1.

2.

5. The Acinetobacter baumannii outer membrane protein liposome vaccine according to claim 4, characterized in that, The administration method of the Acinetobacter baumannii outer membrane protein liposome vaccine is selected from either injection immunization or intranasal immunization.

6. The method for preparing the Acinetobacter baumannii outer membrane protein liposome vaccine according to any one of claims 1-5, characterized in that, include: The outer membrane protein and lipid mixture of Acinetobacter baumannii were dissolved separately using surfactants, and then the outer membrane protein liposomes were prepared by dialysis.

7. The preparation method according to claim 6, characterized in that, The surfactant is selected from any one of N-decanoyl-N-methylglucosamine and octyl-β-D-glucopyranoside.

8. The preparation method according to claim 7, characterized in that, The surfactant is octyl-β-D-glucopyranoside.

9. The preparation method according to claim 6, characterized in that, The preparation method further includes subjecting the obtained outer membrane protein complex liposomes to sonication, centrifugation, and gradient extrusion.

10. The preparation method according to claim 9, characterized in that, The ultrasound is either probe ultrasound or water bath ultrasound.

11. The preparation method according to claim 9, characterized in that, The centrifuge speed is 4000-5500 rpm.

12. The preparation method according to claim 9, characterized in that, The gradient extrusion is performed by sequentially extruding liposomes using 800 nm, 400 nm, and 200 nm carbonate membranes, respectively.

13. The preparation method according to claim 6, characterized in that, The method for preparing the outer membrane protein of Acinetobacter baumannii includes: ligating the nucleotide sequence encoding the antigen to an expression vector to obtain a recombinant expression vector, then transforming it into a host bacterium, culturing and inducing the recombinant bacterium to express the outer membrane protein, and then obtaining the recombinant protein after separation and purification; the antigen is a combination of Omp38 and BauA, or a combination of Omp38, BauA and FimA.

14. The preparation method according to claim 13, characterized in that, The expression vector includes pET-30a; the host bacterium is Escherichia coli BL21(DE3).

15. The preparation method according to claim 13, characterized in that, The purification process includes affinity chromatography of the crude product.

16. The preparation method according to claim 15, characterized in that, The packing material used in the affinity chromatography is Ni-NTA, and the eluent is a urea solution.

17. The use of the Acinetobacter baumannii outer membrane protein liposome vaccine as described in any one of claims 1-5 or the Acinetobacter baumannii outer membrane protein liposome vaccine obtained by the preparation method as described in any one of claims 6-16 in the preparation of a medicament for the prevention of Acinetobacter baumannii infection.

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