Efficient Immune Shield: Biomimetic Nanovaccine of Acinetobacter Baumannii Recombinant Outer Membrane Protein W

Efficient Immune Shield: Biomimetic Nanovaccine of Acinetobacter Baumannii Recombinant Outer Membrane Protein W | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficient Immune Shield: Biomimetic Nanovaccine of Acinetobacter Baumannii Recombinant Outer Membrane Protein W Chenghua Zhu, Shuaiyuan Liang, Ning Yang, Shan Li, Jianpeng Xue, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5642310/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The development of vaccines is an effective and safe strategy to combat multidrug-resistant (MDR) Acinetobacter baumannii ( A. baumannii ) infections. This study aimed to prepare a DC-targeting multiepitope peptide biomimetic nanovaccine and evaluate its immune response and protective effect in mice. Results The B-cell and T-cell epitopes of the OmpW protein from A. baumannii were predicted and screened using bioinformatics methods and identified by immunological means. The selected dominant epitopes were conjugated in series with 6-aminocaproic acid, and a multiepitope peptide, rOmpW, was chemically synthesized. Then, rOmpW was encapsulated with polylactic-co-glycolic acid (PLGA) and a neutrophil membrane (NM), and the surface was modified with DC-targeting peptide (DCpep) to construct the biomimetic nanovaccine DCpep-NM-PLGA-rOmpW. This biomimetic nanovaccinecan induce strong Th1 and Th17 cellular immune responses and humoral immunity. The biomimetic nanovaccine produced efficient immunological protection in an acute lethal pneumonia model of A. baumannii . Conclusions Our results indicate the potential translational value of this biomimetic nanovaccinefor preventing A.baumannii infection. Acinetobacter baumannii biomimetic nanovaccine PLGA neutrophil membrane DCpep Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Acinetobacter baumannii ( A. baumannii ) is a gram-negative opportunistic pathogen and a common pathogen of hospital-acquired infections. The widespread emergence of multidrug-resistant (MDR) and pandrug-resistant (PDR) strains poses a significant threat to human health [ 1 , 2 ] . Many scholars have been committed to developing new antibacterial drugs against A. baumannii infections, but in the past 30 years, very few new antibiotics have been developed [ 3 ] . Therefore, finding new treatment methods other than new antibacterial drugs is urgent. Vaccines have been proven safe and effective in preventing many bacterial infections [ 4 ] . However, none of them have yet to be applied in the clinic. Although the initial vaccines developed from whole-inactivated or live-attenuated strains were easy to prepare and had partial immune protection effects, the use of formaldehyde as the primary inactivating agent cannot fully inactivate A. baumannii and poses potential safety concerns [ 5 ] . Some researchers have shown that the outer membrane of A. baumannii contains more antigenic epitopes, such as outer membrane proteins and capsular polysaccharides, but only a few antigens play immune-protective roles [ 6 – 9 ] . These proteins are complicated to extract, have lower purity, and are unsuitable for mass production. OmpW, a highly conserved small molecule hydrophobic outer membrane pore protein in A. baumannii , is involved in the absorption of iron ions, and is related to resistance to carbapenem antibiotics [ 10 , 11 ] . Some studies have shown that OmpW has active and passive immunoprotective effects on A. baumannii- infected mice, but it significantly inhibits the proliferation of 293T and A549 cells [ 12 , 13 ] . To address this shortcoming, we used bioinformatics software and immunological methods to predict and screen for optimal T-cell and B-cell epitopes on the A. baumannii OmpW protein in model animals. Finally, we constructed and chemically synthesized the multiepitope peptide rOmpW by concentrating the screened dominant B-cell epitopes and the dominant T-cell epitopes, which preserved the immunogenicity of the protein and avoided harmful effects. Given the protein’s tiny size, lack of tertiary structure, short half-life, and susceptibility to proteolytic enzyme degradation in vivo and the low immunogenicity of the antigenic epitope peptide molecule, adjuvants are needed to enhance the immune response. PLGA is a macromolecular synthetic copolymer formed by the aggregation of glycolic acid and lactic acid, and its degradation products are water and carbon dioxide, which are nontoxic to humans. Due to its excellent biocompatibility, encapsulation, and film properties, it was approved by the United States Food and Drug Administration (FDA) for clinical application in 1989. PLGA is an ideal carrier for epitope vaccines because of its effectiveness in high tissue biodistribution, significant controlled release, and enhancement of antigen presentation and the immune response [ 14 , 15 ] . Nevertheless, the body frequently recognizes and eliminates traditional nanodelivery systems as "nonself" substances, which makes targeted delivery to the lesion challenging. In recent years, a new biomimetic technique for preparing nanoparticles has used cell membrane components on their surfaces [ 16 , 17 ] . Some researchers have used neutrophil membrane (NM) to create drug carriers and biomimetic drug delivery systems. Moreover, some studies have shown that neutrophil membrane-encapsulated nanoparticle-mediated gentamicin can effectively resist drug-resistant Klebsiella pneumoniae infection by preventing immune system clearance and achieving efficient targeted aggregation [ 18 ] . Dendritic cells (DCs) are specialized antigen-presenting cells that play essential roles in innate and acquired immune responses. DCpep is a targeting peptide with 12 amino acids (FYPSYHSTPQRP) that binds to receptors expressed on DCs. It participates in the endocytosis process, promoting immunogens to enter the cell more efficiently without impairing their function [ 19 , 20 ] . In this study, we screened out the dominant B-cell and T-cell antigenic epitopes, encapsulated them with PLGA and NM, surface-modified DCpep, and constructed the A. baumannii biomimetic nanovaccine DCpep-NM-PLGA-rOmpW. Next, we investigated the immune efficacy of the biomimetic nanovaccine in model mice and observed its immunoprotective effect on A. baumannii acute lethal pneumonia models to provide a laboratory basis for the clinical application of the biomimetic nanovaccine and potential candidate strategy for the prevention of A. baumannii infection. Materials and Methods Materials The ATCC17978 strain and the human lung adenocarcinoma cell strain A549 were maintained in our laboratory. PLGA (lactide: glycolide = 50:50; MW = 30,000–60,000), IL-4, IFN-γ, IL-17A, IgG1, and IgG2a ELISA kits; human neutrophil extraction kits; and anti-mouse CD3, IgG, CD19, CD80, CD11c, F4/80, Taq enzyme, dNTP, T4 DNA ligase, BamH Ⅰ, Hind III, Nde I, Hind III and DNA marker antibodies were all purchased from Sigma‒Aldrich (St. Louis, MO). Animals Female BALB/c mice, aged 6–8 weeks, were purchased from Shanghai Super-B&K Laboratory Animal Corp. Ltd. (Shanghai, China) and raised at the Laboratory Animal Center of Nanjing Medical University. All the animal experiments were approved by the Ethics Committee of Nanjing Medical University (Approval No. IACUC-2404066). Expression and Purification of OmpW Specific PCR primers were designed according to the gene sequence encoding the OmpW protein: upstream primer: 5'-AAGGATCCGGGTAATTGGCAAGTAAAATTTG-3', downstream primer: 5'-TACTCGAGTTAGAATTTAGCTATAACC-3'. The following PCR conditions were used: thirty cycles of predenaturation (one minute at 98°C), denaturation (fifteen seconds at 98°C), annealing (thirty seconds at 58°C), and extension (thirty seconds at 72°C), followed by extension (one minute at 72°C) and storage (at 10°C). The target gene and empty plasmid were double digested using the restriction endonucleases BamH I and Xho I and then ligated and transformed into E. coli BL21 (DE3) competent cells. Then, the colonies were picked, the plasmids were extracted, and the target gene was enzymatically digested and identified. IPTG-induced recombinant and target protein expressions were analyzed by 15% SDS‒PAGE. The target recombinant proteins were purified using Ni-NTA affinity chromatography, and the OmpW protein concentration was determined using a bicinchoninic acid (BCA) protein quantification kit after endotoxin was removed from the recombinant protein mixture. Prediction and Identification of B-Cell Epitopes and T-Cell Epitopes of OmpW The B-cell epitopes of the OmpW protein were predicted using the OptimumAntigen Design Tool (GenScript, China) according to their secondary structure, surface accessibility, flexibility, hydrophilicity, and antigenic index. After comprehensive analysis, B-cell candidate epitope peptides with a length of 14 amino acids and no overlap were selected. (Figure S1 A). When predicting T-cell epitopes, the Immune Epitope Database Analysis Resource (IEDB) ( http://tools.iedb.org/mhcii/ ) was used to analyze and predict the A and E subregions of MHC-II genes in mice. (Figure S1 B). T-cell candidate epitopes with a length of 15 amino acids, no overlaps, and higher scores were stored. The predicted B-cell and T-cell epitope peptides were chemically synthesized by GenScript Biotechnology Co. Ltd (China). Twelve BALB/c mice were randomly and equally divided into the OmpW group and the control group. The mice in the OmpW group were subcutaneously injected with 100 µg of whole-length OmpW protein mixed thoroughly with an equal volume of Freund's adjuvant, and the final volume was 100 µl. The mice in the control group were injected with an equal volume of PBS. Both groups were subcutaneously immunized three times at two-week intervals; one week after the final immunization, the specific IgG antibody levels in the serum were detected by indirect ELISA. In addition, spleen cells were isolated, adjusted to a concentration of 1×10 6 cells/ml, and stimulated with 20 µg/ml T-cell antigen epitopes. After 72 h, the supernatant was collected, and the gamma interferon (IFN-γ) level in the supernatant was measured using ELISA. Through analysis, we screened two dominant B-cell antigenic epitopes (amino acid sequences: EQGVADKVKEDFG and AKYHFKNSTRFTPY) and one dominant T-cell epitope (amino acid sequence: PSEDTTTALGVVKAD) of the OmpW protein and constructed the multiepitope peptide rOmpW. Constructed and Chemically Synthesized rOmpW The peptides of two dominant B-cell epitopes (OmpW B1 and B6) and one T-cell epitope (OmpW T4) screened were connected in series by 6-aminohexanoic acid to construct rOmpW with 45 amino acids, synthesized by Gill Biochemistry Co. Ltd. (Shanghai). The molecular weight of rOmpW was 4974.60 Da as determined by mass spectrometry, and the purity of rOmpW was 95.50% as determined by high-performance liquid chromatography (HPLC). Safety of rOmpW in vitro Each 10 µl culture of OmpW (10, 20, 40, or 80 µg/ml) or rOmpW (10, 20, 40, or 80 µg/ml) was incubated with A549 cells for 24 or 48 h, respectively, and the level of A549 cell proliferation was detected by CCK-8 assay. Validity Assessment of rOmpW in vivo Sixty BALB/c mice were divided randomly and equally into five groups: the OmpW, rOmpW (25, 50 and 100 µg), and control groups. 50 µg of whole-length OmpW protein was used in the OmpW group; and 25, 50, and 100 µg rOmpW mixed respectively and thoroughly with an equal volume of Freund's Adjuvant were utilized in the rOmpW groups with a final volume of 100 µl; the control group was given an equal volume of PBS. The mice were subcutaneously injected three times every two weeks. One week after the final immunization, the level of rOmpW specific-IgG antibody in the peripheral blood of the mice was detected by indirect ELISA, and the level of INF-γ secretion in the supernatant of the spleen cells was detected by ELISA. Two weeks after the final immunization, a lethal dose of the ATCC17978 (2×10 8 CFU) strain was intratracheally administered to each mouse to establish acute lethal pneumonitis models of A. baumannii . After 24 h, the peripheral blood and right lung tissue homogenate were collected for colony culture and counting. The left lung tissue was stained with HE to observe the pathological changes for seven consecutive days, and the survival status, weight changes, and symptom scores of the mice were recorded daily. The symptom scoring of the mice was mainly based on activity status, stooping, shut eyes, fur condition, and movement. Individual symptom scores were assigned as 0 (normal and active), − 1 (partially disorganized fur), − 2 (obviously disorganized fur, slight stooping), − 3 (a large amount of fur loss, obvious stooping, slow movement, shut eyes), − 4 (inability to stand or move), or − 5 (death). Preparation of Human Neutrophil Membranes Blood samples from 10 healthy adults (15 ml/person, 150 ml total) were collected using anticoagulant tubes (approved by the Second Affiliated Hospital of Nanjing Medical University, Approval No. 2023-KY-165-01). Neutrophils were extracted using a peripheral blood neutrophil isolation kit. A Diff-Ouik Stain kit was used to identify the neutrophils. The washed neutrophils were resuspended in hypotonic solution (PBS: ultrapure water = 1:10) and lysed at room temperature for one hour. The lysed cells were subsequently pulverized in an ultrasonic cleaning tank at 4°C for 5 minutes and processed by middle-frequency ultrasonication. The first centrifugation was performed at 3500 rpm/4°C for five minutes, then a second centrifugation was performed at 4°C/14,500 rpm for 40 minutes. To obtain uniformly sized neutrophil membranes, we used a polycarbonate membrane with a pore size of 200 nm and extruded the neutrophil membranes with a miniextruder. Preparation of DC-targeted peptides (DCpep) Nanjing Putao Biotechnology Co. Ltd. was commissioned to chemically synthesize DCpep with 12 amino acids. The molecular weight of DCpep was identified as 1479.63 Da by mass spectrometry, and the purity of the synthesized peptide was 98.23% as determined by HPLC. To modify the cytomembrane using the phospholipid insertion method, we commissioned this Nanjing Putao Biotechnology to prepare DSPE-PEG2000-DCpep. Construction of the Biomimetic Nanovaccine rOmpW encapsulated with PLGA was prepared using a water/oil/water double emulsion evaporation technique. PLGA-rOmpW was mixed with human neutrophil membranes at a ratio of 5:1, after which the mixture was extruded using a miniextruder through a polycarbonate membrane with a pore size of 200 nm. The mixture was centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the mixture was resuspended in PBS to obtain NM-PLGA-rOmpW. DSPE-PEG2000-DCpep was added to the NM-PLGA-rOmpW solution at a ratio of 1:5. The mixture was stirred for two hours in the dark and then centrifuged at 10,000 rpm for 15 min. After that, the supernatant was discarded, and the precipitate was collected. Finally, the A. baumannii biomimetic nanovaccine DCpep-NM-PLGA-rOmpW was obtained by resuspension in PBS buffer. Particle size, PDI, and Zeta Potential Measurement Each 3 ml emulsion of PLGA-rOmpW and DCpep-NM-PLGA-rOmpW was diluted with ultrapure water in moderation and placed in a cuvette. The average particle size, polydispersity index (PDI), and zeta potential were measured by a particle size analyser. Each sample was measured five times, and the average value was taken to ensure the accuracy of the results. Membrane protein properties of DCpep-NM-PLGA We performed polyacrylamide gel electrophoresis (SDS-PAGE) to validate the presence of neutrophil membrane proteins on the surface of DCpep-NM-PLGA. Each 20 µl solution of PLGA, NM, NM-PLGA, or DCpep-NM-PLGA was added to four electrophoretic channels. After the electrophoresis was completed, Coomassie Brilliant Blue solution was added to stain the gels for 30 minutes, followed by one hour of washing. The gels were then photographed and the images were recorded. Transmission Electron Microscopy (TEM) To observe the morphology of the neutrophil membranes, PLGA, NM-PLGA and DCpep-NM-PLGA, the four substances were diluted 20 times with ultrapure water. A small amount of each dilution liquid was subsequently dropped on a copper grid and left for 2 minutes to allow the liquid to permeate naturally. Next, the excess liquid was gently removed from the surface of the copper grid with filter paper to ensure that it was dried naturally at room temperature. After drying, negative staining was performed with 0.1% phosphotungstic acid for 5 min, and the samples were subjected to transmission electron microscopy (TEM) to observe the microscopic morphology in detail. Colocalization in cells We commissioned China Pharmaceutical University to use PLGA to encapsulate Cyanine-3 (CY3), which is entirely insoluble in water and soluble in chloroform. The neutrophil membranes were incubated with DIL for 20 min in the dark and then centrifuged at 1500 rpm for 5 min, after which the precipitate was collected and resuspended in PBS. We mixed 0.5 mg of PLGA with 0.1 mg of neutrophil membranes, selected a PVDF membrane with a pore size of 200 nm, and physically extruded the mixture using a small mini extruder. Finally, we obtained the NM-PLGA reference for the phospholipid insertion method. FITC-DSPE-PEG2000-DCpep (0.1 mg) and 0.5 mg of NM-PLGA were stirred away from light for one hour and then centrifuged at 10,000 rpm for 5 min, after which the precipitate was removed and resuspended in PBS to obtain DCpep-NM-PLGA. Each 50 µl solution of PLGA, NM-PLGA, or DCpep-NM-PLGA was coincubated with RAW264.7 cells for one hour. The colocalization phenomenon in cells was observed using laser scanning confocal microscopy. Detection of the Encapsulation Efficiency A standard curve was generated to measure the encapsulation rate of rOmpW in the A. baumannii biomimetic nanovaccine, and the amount of unencapsulated rOmpW was indirectly calculated by HPLC. Finally, the encapsulation rate (EE) was calculated. Encapsulation rate (%) = (total rOmpW input - rOmpW amount in the supernatant)/total rOmpW input × 100. In vitro Peptide Release Five milliliters of DCpep-NM-PLGA-rOmpW was placed in a shaker at 37°C and 100 rpm to simulate the release process under physiological conditions. The release of the rOmpW peptide was determined at various time intervals (1, 2, 4, 8, 13, 24, 30, 36, 48, 54, 60, and 72 h). After the samples were centrifuged at high speed, the supernatants were collected, and the rOmpW peptide concentration was accurately determined by HPLC. Safety in vitro A549 cells were coincubated with 10 µL of PBS (as the control group) or different concentrations of DCpep-NM-PLGA-rOmpW (10, 20, 40, or 80 µg/ml rOmpW) for 24 or 48 h. The effects of these combinations on the proliferation of A549 cells were detected by CCK-8 assay. Establishment of the grouping, immunization and modeling of mice Sixty BALB/c mice were randomly and equally divided into four groups: the DCpep-NM-PLGA-rOmpW, DCpep-NM-PLGA-PBS, rOmpW and NS groups. Each group was immunized with 200 µl of DCpep-NM-PLGA-rOmpW containing 50 µg of rOmpW, 200 µl of DCpep-NM-PLGA-PBS, 50 µg of rOmpW dissolved in 100 µl of NS with 100 µl of Freund’s adjuvant, and 200 µl of saline. All the mice were subcutaneously immunized every two weeks for a total of 3 times. One week after the last immunization, the rOmpW-specific antibody level in the peripheral blood of the mice was detected by indirect ELISA, and the secretion levels of IgG1 and IgG2a in the serum and IL-4, IL-17A, and INF-γ in the supernatant of the spleen cells were detected by ELISA. Flow cytometry was used to detect the frequencies of CD19 + , CD3 + , CD11c + , and F4/80 + cells in the inguinal lymph nodes. Two weeks after the final immunization, a lethal dose of ATCC17978 was administered via the trachea to establish the acute fatal pneumonia model of A. baumannii . Colony counts in peripheral blood and lung tissue were performed 24 hours later. HE staining was used to observe pathological changes in the left lung tissue. The remaining mice were monitored for one week, and their survival status, weight changes, and clinical symptom scores were recorded daily. Specific Antibody Detection of rOmpW The rOmpW was first diluted to 10 µg/mL to coat a 96-well plate. Then, the resulting mixture was added to each well (100 µL per well) and incubated at 4°C for 12–18 h. After washing five times with PBS and 0.05% Tween 20 (PBST), 200 µL of 2% bovine serum albumin (BSA) was added to each well and incubated at 37°C for 2 h. Then, the mouse serum was diluted with PBS at a ratio of 1:200, and 100 µL was added to each well of the OmpW-immunized and PBS groups. The plates were washed five times after incubation at 37°C for 2 h. After that, 100 µL of freshly diluted goat anti-mouse IgG (diluted 1:10000) and horseradish peroxidase (HRP) were added to each well, and the plates were incubated at 37°C for 1 h. The plates were then washed five times, and 100 µL of tetramethylbenzidine solution (TMB) substrate was added to each well in the dark for 20 min. Finally, 50 µL of termination solution was added to each well, and the data were read at an OD of 450 nm. Cytokine Detection One week after the final immunization, the peripheral blood of the mice was collected. The expression levels of IgG1 and IgG2a antibodies in the serum were detected using IgG1 and IgG2a ELASA kits. Splenocytes were isolated and adjusted to a density of 1×10 6 cells/mL. Two hundred microlitres of cell suspension was added to each well of a 96-well plate, the cells were stimulated with rOmpW (20 µg/mL), and the supernatant was collected after 72 h. The levels of IFN-γ, IL-4, and IL-17A were detected using IFN-γ, IL-4, and IL-17A ELISA kits. Flow Cytometric Analysis One week after the final immunization, single-cell suspensions of inguinal lymph nodes were prepared. The cells (1×10 6 /mL) were blocked with Fc blocking antibody for 15 min at 4°C. The cells were washed and stained with fluorescent-conjugated antibodies against lymphocyte surface receptors, APC-Mouse I-Ab, PerCP-Cy5.5-CD11c, PE-F4/80, PE-CD3 and APC-CD19 (BD Biosciences) at 4°C and protected from light for 30 min. Then, the cells were washed and fixed with 2% paraformaldehyde solution for 20 min. Data were acquired on a BD FACS Canto II flow cytometer (BD Biosciences) with at least 1 × 10 5 events for each sample and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). HE staining of lung tissue Twenty-four hours after mice were challenged with A. baumannii ATCC17978, the lungs were removed under aseptic conditions and fixed in 4% formalin. After being stained with haematoxylin‒eosin (HE), the paraffin-embedded sections were examined histopathologically by microscopy. Using a macro in ImagePro software, lung injury was estimated by the percentage of the lesion area among the total lung area. Statistical Analysis Statistical analyses were performed using Statistical Package of Social Sciences (SPSS, version 25.0, SPSS Inc,Chicago,IL). T-test was chosen to compare the mean values between the two samples. One-way analysis of variance (ANOVA) was used to compare multiple variances and determine the homogeneity of variance analysis. P < 0.05 indicated that the difference was statistically significant. Results Expression and Identification of the OmpW Protein The recombinant plasmid pET28a-OmpW was verified by PCR amplification and digestion, and the results of the sequencing analysis are shown in Figure S2. The constructed recombinant plasmid pET-28a-OmpW was transformed into Escherichia coli ( E. coli ) BL21 cells, which were subsequently induced with IPTG. The OmpW protein was expressed in the form of an inclusion body, and the final concentration of the OmpW protein was 1.8 mg/mL, as measured by protein quantitative electrophoresis. The results of SDS‒PAGE revealed a specific protein band at 23 kDa, which was consistent with the expected size of the OmpW protein (Fig. 1 B). Epitopes Prediction and Construction of the Multiepitope peptide rOmpW Through bioinformatics analysis, six B-cell candidate epitope peptides (named OmpW B1–B6) and four T-cell candidate epitope peptides (named OmpW T1–T4) (Table 1 ) were predicted and chemically synthesized. BALB/c mice were subcutaneously injected with 100 µg of recombinant OmpW (1 µg/µL in PBS) or an equivalent volume of PBS three times every two weeks. The vaccination schedule is shown in Fig. 1 A. The peripheral blood and spleen cells were collected one week after the final immunization. OmpW B1 epitopes, B6 epitopes and full-length proteins showed significant antigen-antibody reactions with the serum of the OmpW-immunized group (Fig. 1 C), and the T4 peptide significantly increased the level of IFN-γ secreted by the splenocytes of OmpW-immunized mice (Fig. 1 D). The screened peptides of two dominant B-cell epitopes (OmpW B1 and B6) and one T-cell epitope (OmpW T4) were connected in series using 6-aminohexanoic acid to design and generate a novel multiepitope peptide, rOmpW. rOmpW, with 45 amino acids and a molecular weight of 4974.60 Da, was identified by mass spectrometry (Figure S3A) and HPLC (Figure S3B). Immunoprotective Effects Of rOmpW BALB/c mice were subcutaneously injected with OmpW (50 µg), rOmpW (25, 50, or 100 µg), or an equal volume of PBS three times every two weeks. The peripheral blood and spleens were collected one week after the final immunization. The specific IgG antibody levels in the serum were detected by indirect ELISA. The OD450 nm values of the OmpW and rOmpW groups were significantly greater than those of the control group (P < 0.001), and the OD450 nm values of the 50 and 100 µg rOmpW groups were considerably greater than those of the OmpW and 25 µg rOmpW groups (P < 0.01) (Fig. 2 A). IFN-γ in the supernatant secreted by splenocytes was detected by ELISA. The splenocytes from the OmpW and rOmpW groups produced higher levels of IFN-γ than those from the control group did (***P < 0.001, **P < 0.01), and the level of IFN-γ in the 50 µg rOmpW group was significantly greater than that in the 25 µg rOmpW group (P < 0.05), whereas there was no statistically significant difference from that in the 100 µg rOmpW group (Fig. 2 B). The evaluation of the lethal dose of A. baumannii ATCC17978 is shown in Figure S4. When 100 µl of A. baumannii ATCC17978 (2×10 9 CFU/ml) was injected into the trachea, the 3-day survival rate was 0%. For the subsequent experiments, we selected 100 µl of A. baumannii ATCC17978 (2×10 9 CFU/ml) administered via intratracheal injection to establish an acute fatal pneumonia model. To determine whether rOmp22 conferred protection, we assessed its effectiveness in an acute pneumonia model in BALB/c mice. Two weeks after the last boost immunization, all the groups were challenged with lethal doses of A. baumannii ATCC17978 (2×10 8 CFU/ml). The number of CFUs was counted in the peripheral blood and tissue from the right lung at 24 h post challenge (Fig. 2 C-D). Compared with control mice, OmpW and rOmpW immunized mice presented lower bacterial loads in the blood and lung tissue (***P < 0.001, **P < 0.01, *P < 0.05). The colony counts of the blood and lung tissue in the rOmpW 50 µg group were lower than those in the OmpW and rOmpW 25 µg groups (**P < 0.01, *P < 0.05), and there was no statistically significant difference from the rOmpW 100 µg group. After 24 hours, the left lung tissue was stained with HE and analysed semiquantitatively using Image-Pro Plus software (Fig. 3 E), and the areas of inflammation in the OmpW and rOmpW groups were significantly lower than that in the control group (P < 0.001). In addition, the area of inflammation in the 50 µg rOmpW group was smaller than that in the 25 µg rOmpW group (P 0.05). Six mice challenged with A. baumannii were randomly selected for recording survival, body weight changes and clinical scores every day for seven days (Figure S5A‒C). The survival rates in the rOmpW 25 µg, rOmpW 50 µg, rOmpW 100 µg, and OmpW groups were 50% (3/6), 66.67% (4/6), and 66.67% (4/6), respectively. A survival rate of 50% (3/6) was significantly greater than that in the control group (3-day survival rate of 0). The body weights of the mice in each group decreased to the lowest levels on approximately the second day; after that, the body weights of the surviving mice in the OmpW and rOmpW groups gradually increased and then recovered to the level observed before the challenge on the 6th to 7th days. The clinical scores of the mice reached the lowest level on the second day after the challenge, and the mice in the control group died within three days. The clinical scores of the surviving mice in the OmpW and rOmpW groups gradually increased, and the symptom scores of the surviving mice generally returned to 0 points on the sixth day of observation. Effects of rOmpW on the Proliferation Level of A549 cells We used the CCK-8 method to detect the proliferation level of A549 cells to evaluate the in vitro safety of rOmpW (Fig. 3 A-B). Different concentrations of rOmpW were cocultivated with A549 cells for 24 and 48 hours, and there was no significant inhibitory effect on the growth of A549 cells, whereas OmpW significantly inhibited the growth of A549 cells at a concentration of 20 µg/ml. Preparation of Neutrophil Membranes and DCpep Peripheral blood neutrophils from healthy adults were extracted using a Peripheral Blood Neutrophil Isolate Kit and identified using a CDiff-Quik Stain Kit (Figure S6). Neutrophil membranes were obtained by ultracentrifugation. DCpep, with 12 amino acids and a molecular weight of 1479.63 Da, was chemically synthesized and identified by mass spectrometry (Figure S7A) and HPLC (Figure S7B). Characterization and Identification of Biomimetic Nanoparticles The average particle size of DCpep-NM-PLGA-rOmpW was 135.67 ± 11.26 nm(Fig. 4 A), the PDI was 28.33 ± 4.50%, and the zeta potential was − 10.37 ± 0.17 mV (Fig. 4 B). SDS‒PAGE (Fig. 4 C) revealed that DCpep-NM-PLGA, NM-PLGA, and NM exhibited similar electrophoretic bands, indicating that DCpep-NM-PLGA prepared by extrusion could better maintain the integrity of the protein on the surface of the neutrophil membrane. We modified DCpep with FITC, and FITC-DCpep resulted in an obvious absorption peak at 495 nm in the UV–vis spectrum. The aqueous solution of FITC-DCpep-NM-PLGA also showed an obvious absorption peak at 495 nm (Fig. 4 D), which indicated that DCpep could be successfully modified on the surface of human neutrophil membranes. Transmission electron microscopy (TEM) was performed to assess the morphology and size of the biomimetic nanoparticles. PLGA, NM, and DCpep-NM-PLGA were analyzed by TEM, and the results revealed that the neutrophil membranes could be better attached to the surface of PLGA without affecting the size of the particles and that DCpep-NM-PLGA exhibited spherical structures that were uniform and well dispersed (Fig. 4 E). Fifty microlitres of PLGA, NM-PLGA, or FITC-DCpep-NM-PLGA was cocultured with RAW264.7 cells. After 1 h of incubation, the samples were observed by laser scanning confocal microscopy. Significant colocalization phenomena among DCpep, neutrophil membranes and PLGA could be clearly observed (Fig. 4 F). Encapsulation Efficiency and Release Studies in vitro The standard curve was constructed by HPLC with the rOmpW concentration as the horizontal coordinate and the peak area at the corresponding concentration as the vertical coordinate. The equation of this standard curve was y = 11385x − 217.23, and the R² value was 0.9998, indicating a good linear relationship (Fig. 5 A). The encapsulation efficiency (EE) of DCpep-NM-PLGA-rOmpW was indirectly calculated to be 43.25% by HPLC, which is in line with expectations. DCpep-NM-PLGA-rOmpW was dissolved in 5 ml of physiological saline solution (pH 7.4) in a shaker at 37°C and 100 rpm. The rOmpW content in the supernatant was determined by HPLC. The cumulative release rates of rOmpW from DCpep-NM-PLGA-rOmpW at 1, 2, 4, 8, 13, 24, 48 and 72 h were 17.7%, 23.3%, 27.7%, 32.7%, 33.8%, 34.3%, 38.8% and 44.3%, respectively (Fig. 5 B), revealing a significant burst release of rOmpW at 1 h, followed by a gradual and slow release that could be sustained for 72 h, indicating that the biomimetic nanovaccine had a longer protection time. Immunization Efficacy of the Biomimetic Nanovaccine BALB/c mice were immunized subcutaneously with DCpep-NM-PLGA-rOmpW containing 50 µg/200 µl of rOmpW, an equal volume of DCpep-NM-PLGA-PBS, 50 µg of rOmpW or an equal volume of saline every two weeks for a total of three immunizations. The peripheral blood and spleens were collected one week after the last vaccination. The levels of rOmpW-specific IgG (Fig. 6 A), IgG1 (Fig. 6 B), and IgG2a (Fig. 6 C) in the serum were detected by ELISA. Compared with those in the NS, rOmpW, and DCpep-NM-PLGA-PBS groups, the expression levels of specific IgG, IgG1, and IgG2 antibodies in the serum of the mice in the biomimetic nanovaccine group were significantly greater, suggesting that the biomimetic nanovaccine could induce a stronger humoral immune response. The secretion levels of IFN-γ and IL-17A in the A. baumannii nanovaccine group were also significantly greater (P < 0.001) than those in the NS, rOmpW, and DCpep-NM-PLGA-PBS groups, and the expression levels of IFN-γ, IL-4, and IL-17A in the supernatants of the spleen cells were detected by ELISA (Fig. 6 D-F). However, the expression level of IL-4 in spleen cells was significantly decreased (P < 0.001), suggesting that the biomimetic nanovaccine induced stronger Th1 and Th17 cellular immune responses. One week after the final immunization, flow cytometry was used to detect the percentages of CD19 + , CD3 + , CD11c + , and F4/80 + cells in the single-cell suspensions of the inguinal lymph nodes in each group (Fig. 7 A-F). The frequencies of CD3 + T lymphocytes, CD19 + B lymphocytes, CD11c + DCs, and F4/80 + macrophages in the biomimetic nanovaccine group were significantly greater than those in the NS, rOmpW and DCpep-NM-PLGA-PBS groups (P < 0.001). These findings indicated that the biomimetic nanovaccine could stimulate the differentiation of B and T lymphocytes in the lymph nodes and promote the migration of DCs and macrophages to the lymph nodes. Immunoprotective Effects of Biomimetic Nanovaccine To determine whether the biomimetic nanovaccine conferred immune protection, we assessed its effectiveness in an acute pneumonia model in BALB/c mice. All the groups were challenged with lethal doses of ATCC17978 via the trachea. The bacterial loads in the peripheral blood and lung tissue of the mice immunized with the biomimetic nanovaccine were lower than those in the peripheral blood and lung tissue of the mice in the other three groups 24 h postchallenge (Fig. 8 A-B). The survival status, weight changes, and clinical scores of the mice in each group were recorded for seven consecutive days after the A. baumannii ATCC17978 challenge. The one-week survival rates were 83.33% (5/6) in the biomimetic nanovaccine group and 66.67% (4/6) in the rOmpW group, whereas all the mice in the NS and DCpep-NM-PLGA-PBS groups died within three days (Fig. 8 C). The weight of the mice in each group decreased to the lowest level on approximately the second day after A. baumannii challenge, and the weights of the surviving mice in the biomimetic nanovaccine and rOmpW groups gradually increased and generally recovered to the preattack levels on approximately the seventh day (Fig. 8 D). Symptom scores decreased to the lowest level in each group on the 2–3 days after A. baumannii challenge. The symptom scores of surviving mice in the biomimetic nanovaccine and the rOmpW groups gradually increased and generally returned to normal levels on the seventh day (Fig. 8 E). HE staining was used to observe pathological changes in the left lung tissue (Fig. 8 F), and the results revealed substantial inflammatory cell infiltration (mainly neutrophilic), necrosis of the lung tissue and damage to the structure of the alveolar lumen in the NS and DCpep-NM-PLGA-PBS groups. Moreover, the lung tissue in the rOmpW group was infiltrated by a few inflammatory cells, the interstitial lung tissue was thickened slightly, and the structure of the alveolar lumen was intact. However, in the biomimetic nanovaccine group, although a small number of inflammatory cells infiltrated the lung tissue, there was no edema or thickening in the interstitium of the lung tissue. The Image-Pro Plus software analysis results revealed that the inflammatory area of the lung tissue in the biomimetic nanovaccine group was significantly lower than those in the other three groups (each P < 0.001). Duration of rOmpW-Specific IgG antibodies in serum The specific IgG level in the serum was detected by indirect ELISA at five time points: 1, 3, 5, 7, and 9 weeks after the final immunization (Fig. 9 ), and venous blood was collected from the inner canthal orbital vein of each mouse. The OD value of the serum-specific IgG in the biomimetic nanovaccine group was significantly higher than those in the other three groups at all five time points (P < 0.001). In the ninth week, the serum-specific IgG level in the biomimetic nanovaccine group remained high. In contrast, the serum-specific IgG level in the rOmpW group gradually decreased in the 5th week and was not significantly different from that in the NS group at the 9th week (P > 0.05). Safety of the Biomimetic Nanovaccine Toxicity is a primary concern when vaccines are used in biomedical applications. We tested the toxicity of the biomimetic nanovaccine in A549 human lung adenocarcinoma epithelial cells and in mice. The results showed that the biomimetic nanovaccine had no inhibitory effect on A549 cells (Figure S8A-B). One week after the final immunization, the serum and major organs of the mice were collected to detect changes in the serum levels of BUN, Cre, ALT, and AST. Histological changes in the heart, liver, spleen, lung, and kidney were assessed by HE staining (Figure S8C-G). The biomimetic nanovaccine caused no apparent damage to liver or kidney functions or the pathology of major organs (heart, liver, spleen, lungs, and kidneys). These results indicate that the biomimetic nanovaccine has a preeminent safety profile. Discussion Acinetobacter baumannii is a strictly aerobic gram-negative bacillus that is highly resistant to humidity, ultraviolet radiation, and chemical disinfectants and can cause pneumonia, meningitis, urinary tract infections, bloodstream infections, and other disorders [ 1 ] . The resistance mechanism of A. baumannii is complicated, and it is resistant to all commonly used clinical antibiotics. The development of new antimicrobial drugs against A. baumannii takes a long time and also tends to induce new resistance in A. baumannii . Vaccines are effective ways to prevent and control A. baumannii infection. The development of vaccines against A. baumannii has become a hot topic for many researchers [ 4 ] . OmpW is an important outer membrane protein of A. baumannii . Hypoxia can lead to the downregulation of OmpW expression, resulting in decreased adhesion and invasion of A. baumannii to human lung epithelial cells and reduced biofilm formation by A. baumannii [ 13 ] . The OmpW protein is highly conserved among A. baumannii strains. The homology of OmpW is more than 91% among 804 A. baumannii strains. OmpW significantly reduces bacterial loads in various organs in A. baumannii infection models and improves survival in mice. It also has passive immunoprotective benefits. However, OmpW has a slight inhibitory effect on the proliferation of 293T and A549 cells, suggesting that OmpW may have some toxicity [ 12 ] . In this study, the dominant antigenic epitopes of OmpW were predicted using bioinformatics methods and screened out by immunological means in vivo. The selected dominant antigenic epitopes were concatenated with 6-aminohexanoic acid and chemically synthesized to construct a multiepitope peptide, rOmpW. rOmpW was verified to have no inhibitory effect on the growth of A549 cells in vitro and to have an immunoprotective impact in vivo. The results showed that rOmpW induced stronger humoral and cellular immune responses in mice and increased their productivity. When the immunization dose of rOmpW was 50 µg, the levels of specific IgG in the serum and Th1 cytokines (IFN-γ) in the supernatant secreted by the spleen cells were significantly greater than those in the OmpW and rOmpW 25 µg groups, and there was no significant difference from those in the rOmpW 100 µg group. Therefore, 50 µg of rOmpW was considered an appropriate dose for a single immunization, which laid the foundation for subsequent experiments. Due to the small size of the rOmpW segments, they are easily degraded by proteases in vivo, and their immunogenicity is relatively weak. To overcome these limitations and enhance the immunogenicity of rOmpW, we used PLGA as an adjuvant and carrier. However, conventional nano delivery systems are often cleared by the body as "not self", and the direct fusion of cell membranes with nanoparticles, a new method developed in recent years, reduces the clearance of the nanoparticles by the immune system. Neutrophil membranes have been proven to offer the advantages of immune escape and specific targeting in infections and tumors, among other effects [ 21 – 23 ] . In this study, we extracted neutrophils from the peripheral blood of healthy adults and successfully prepared neutrophil membranes by ultracentrifugation. To reduce immune clearance, biomimetic modification of PLGA-rOmpW with neutrophil membranes was performed. Due to their strong antigen-presenting ability, presenting dendritic cells (DCs) play a crucial role in natural and acquired immune responses to pathogens. The molecules targeted by DCs are essential in developing targeted drugs and vaccines for DCs. Dendritic cell-targeting peptide (DCpep) specifically binds to the receptors expressed on DCs, promoting the migration and activation of DCs, enhancing the delivery of immunogens to T and B lymphocytes, and activating Th1, Th2, and Th17 immune responses [ 24 , 25 ] . In this study, DCpep-modified NM-PLGA was used for the first time to prepare a biomimetic nanovaccine. We successfully prepared PLGA-rOmpW using a water/oil/water double emulsion evaporation technique and obtained NM-PLGA-rOmpW by physical extrusion. By applying the phospholipid insertion method, we successfully prepared DCpep-NM-PLGA-rOmpW by surface modification of DCpep [ 26 ] . The characterization of DCpep-NM-PLGA-rOmpW revealed that the particle size was less than 200 nm, with a smooth spherical structure, uniform particle size, and good dispersion, as determined by transmission electron microscopy. In this study, we observed the release characteristics of DCpep-NM-PLGA-rOmpW in vitro, and the results revealed that rOmpW presented a biphasic pattern of initial burst release followed by sustained release in vitro . Early rapid release can trigger a robust immune response. In contrast, the later sustained and slow release could continuously stimulate the body to produce antibodies and related cytokines, thus enhancing the immune response. Safety is the key to whether the nanovaccine can be used in vivo . Therefore, we evaluated the safety of the biomimetic nanovaccine in vitro and in vivo . We found that it had no inhibitory effect on the proliferation of A549 cells and did not damage liver or kidney functions or important organs, indicating that the biomimetic nanovaccine is safe and non-toxic and can be used for experimental studies in vivo. To verify the immunoprotective effect of the biomimetic nanovaccine, we examined the levels of the humoral immune response (rOmpW-specific IgG, IgG1, and IgG2a in serum) and the cellular immune response (IFN-γ, IL-4, and IL-17A in the supernatants of splenocytes) in mice. Humoral immunity is one of the most important mechanisms by which the body fights against pathogens, and the levels of specific antibodies in serum and their subtypes constitute an essential basis for evaluating the humoral immune response induced by vaccines [ 27 – 30 ] . This study revealed that the biomimetic nanovaccine could increase the levels of specific IgG, IgG1 and IgG2a in the serum of mice. CD4 + T cells can be differentiated into Th1, Th2 and Th17 subpopulations. Th1 cells mainly secrete cytokines such as IL-2 and IFN-γ to mediate the cellular immune response. Th17 cells secrete IL-17A, which can induce the production of many chemokines, facilitate the recruitment of neutrophils, and play a key role in bacterial clearance [ 31 – 33 ] . We found that the biomimetic nanovaccine could induce stronger Th1 (IFN-γ) and Th17 (IL-17A) immune responses. In addition, antibody duration is also an important indicator for evaluating vaccine effects [ 34 ] . We found that the level of the rOmpW-specific IgG antibody induced by the biomimetic nanovaccine could be maintained more persistently, suggesting that the biomimetic nanovaccine may have a more prolonged immunoprotective effect. Moreover, flow cytometry revealed significant increases in the frequencies of CD11 + DCs, F4/80 + macrophages, CD3 + T lymphocytes and CD19 + B lymphocytes in the lymph nodes, suggesting that part of the mechanism of the immune response induced by the biomimetic nanovaccine may be related to promoting the migration of DCs and macrophages to the lymph nodes and stimulating the differentiation of B and T lymphocytes. Establishing an infection model is a key step in evaluating the protective effect of vaccines [ 35 ] . We constructed an acute lethal pneumonia model of A. baumannii infection by injecting a lethal dose of a standard A. baumannii strain into the trachea in mice. We found that the biomimetic nanovaccine significantly reduced the bacterial load in the lung tissue and blood, improved the symptom scores, and reduced the mortality rate of the mice in the acute lethal pneumonia model. These findings indicate that the biomimetic nanovaccine DCpep-NM-PLGA-rOmpW can exert effective immunoprotection. Conclusion In summary, a novel biomimetic nanovaccine, DCpep-NM-PLGA-rOmpW, was prepared. This biomimetic nanovaccine could promote the migration of DCs and macrophages to the lymph nodes, induce the proliferation of B and T lymphocytes, enhance the humoral immune response and Th1 and Th17 cellular immune response, and exert a better immunoprotective benefit. Therefore, this biomimetic nanovaccine is a desirable candidate vaccine for preventing A. baumannii infection. Declarations Acknowledgements The authors gratefully acknowledge the contributions of Dr. Naveen Nagiah, a former postdoc at IMSTEL, for initiating this study. We also extend our appreciation to Dr. Sylvia L. Natividad and her student, Jose Perez, for their assistance in acquiring the videos. Furthermore, we acknowledge the support provided by the NIH-NIMHD-RCMI Grant no. 5G12MD007592, which facilitated the utilization of the confocal microscopy facility in BBRC at UTEP. Authors Contributions IH and GC: Data curation, Formal Analysis, Investigation, Methodology, Project administration, Software, Visualization, Validation, Writing – Original draft, Writing – review & editing; AEP: Data curation, Formal analysis, Investigation, Software, Visualization, Writing – review & editing. CDL: Conceptualization, Investigation, Methodology, Project administration, Writing – review & editing; BJ: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Visualization, Resources, Supervision, Validation, Writing – Original draft, Writing – review & editing. Funding This research was supported by grants from the National Institutes of Health (NIH 1SC1HL154511-01) and the National Science Foundation (NSF 1927628) awarded to BJ. Data Availability The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. Competing Interests : The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported. However, it is important to note that BJ is a member of the editorial board of this journal. This relationship has been disclosed to ensure transparency in the review process. References Dandachi I, Azar E, Hamouch R, Maliha P, Abdallah S, Kanaan E, Badawi R, Khairallah T, Matar GM, Daoud Z. Acinetobacter spp in a Third World Country with Socio-economic and Immigrants Challenges. J Infect Dev Ctries. 2019;13(11):948–55. Holmes AH, Moore LS, Sundsfjord A, Steinbakk M, Regmi S, Karkey A, Guerin PJ, Piddock LJ. Understanding the mechanisms and drivers of antimicrobial resistance. Lancet. 2016;387(10014):176–87. 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Duration of humoral immunity from smallpox vaccination and its cross-reaction with Mpox virus. Signal Transduct Target Ther. 2023;8:350. Chuang YC, Cheng A, Sun HY, Wang JT, Chen YC, Sheng WH, Chang SC. Microbiological and clinical characteristics of Acinetobacter baumannii bacteremia: Implications of sequence type for prognosis. J Infect. 2019;78(2):106–12. Tables Table 1 Location and sequence of OmpW B-cell and T-cell antigen epitopes. Epitopes Location Amino acid sequence OmpW B1 124–137 EQGVADKVKEDFGV OmpW B2 80–93 LLDGQKVARIKQLP OmpW B3 161–174 YADISPEVTLTNGA OmpW B4 34–47 SEDTTTALGVVKAD OmpW B5 147–160 PADAKNWGVFVDVR OmpW B6 98–111 AKYHFKNSTRFTPY OmpW T1 20–34 GNWQVKFGGSVIAPS OmpW T2 46–60 ADHEYAFTPSVEYFF OmpW T3 105–119 STRFTPYIGIGATAF OmpW T4 33–47 PSEDTTTALGVVKAD Additional Declarations No competing interests reported. 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University","correspondingAuthor":false,"prefix":"","firstName":"Jianling","middleName":"","lastName":"Huang","suffix":""},{"id":391822324,"identity":"efdd67c5-e052-4553-b820-06983acd8df5","order_by":11,"name":"Ganzhu Feng","email":"","orcid":"","institution":"Second Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ganzhu","middleName":"","lastName":"Feng","suffix":""},{"id":391822325,"identity":"275826dd-9098-4ab0-aad1-470dfa92fcba","order_by":12,"name":"Xingran Du","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDACZgjF2MDewGAAZh4gWgvPAWK1MMC0SCRAmYS0GBznPfy6ouaO7Iabbw8U3WxjkOO7kcD4uQCPFslmvjTLM8eeGW+4nZdgnNvGYCx5I4FZegYeLfzMPGaGDWyHEzfczjEAaUnccCOBjZkHjxY2sJZ/QC03z4C11BPUArTF+GFjG1DLDR6wlgQDQlokm3nMGBv7DhvPPAN0WM45CcOZZx42S+PTYnD+jPHHhm+HZfuOnzEzzimzkec7nnzwMz4tIO9IwBjAqASxGRvwawDG/wcY4wEhpaNgFIyCUTAyAQA7tU2lFNvRAwAAAABJRU5ErkJggg==","orcid":"","institution":"The Affiliated Jiangning Hospital with Nanjing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xingran","middleName":"","lastName":"Du","suffix":""}],"badges":[],"createdAt":"2024-12-14 08:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5642310/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5642310/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71985769,"identity":"cba88e31-d4e9-496a-ba02-c862be1e84da","added_by":"auto","created_at":"2024-12-20 10:45:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1023241,"visible":true,"origin":"","legend":"\u003cp\u003eEpitope prediction and design of the multiepitope peptide rOmpW.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes:\u003c/strong\u003e (A) Mouse immunization scheme. (B) SDS‒PAGE of purified recombinant OmpW protein. Lane 1: OmpW, 1 μl; Lane 2: OmpW, 2 μl; Lane 3: Bovine serum albumin (BSA), 1 μg; Lane 4: BSA, 2 μg; Lane 5: BSA, 4 μg. (C) Indirect ELISA test of candidate B-cell epitopes and OmpW-immunized mouse serum. (D) IFN-γ levels in the splenocyte supernatant. The data are expressed as the means ± SDs (n=6), ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5642310/v1/223e6f421f02debcd5c463a8.png"},{"id":71986560,"identity":"d2f43af4-9a41-4717-bf84-8a8288744678","added_by":"auto","created_at":"2024-12-20 10:53:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2179742,"visible":true,"origin":"","legend":"\u003cp\u003eImmunoprotective effects of rOmpW in BALB/c mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes:\u003c/strong\u003e One week after the last immunization, (A) rOmpW-specific IgG at a 1:200 dilution was obtained from the sera of mice in the different groups. (B) IFN-γ concentrations released from splenocyte supernatants in different groups. After \u003cem\u003eA. baumannii\u003c/em\u003e ATCC17978 challenge for 24 h, colony counts in (C) peripheral blood and (D) lung tissue and (E) lung histopathology (200×) were performed for the different groups. The data are presented as the means ± SDs (n=3). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5642310/v1/c8f1516b96c863279e3e2cc5.png"},{"id":71987287,"identity":"d219d873-c064-4aeb-9bc4-d6e15b7c3595","added_by":"auto","created_at":"2024-12-20 11:01:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120621,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of coincubation of A549 cells with different concentrations of OmpW and rOmpW for 24 h (A) and 48 h (B) .\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5642310/v1/df31c4f2f4f7b8bd7acecd17.png"},{"id":71985773,"identity":"3f704e8d-b2f5-4aae-a588-ac375e5fc1f6","added_by":"auto","created_at":"2024-12-20 10:45:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4465873,"visible":true,"origin":"","legend":"\u003cp\u003ePhysical–structural characterization of the biomimetic nanoparticles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes:\u003c/strong\u003e Size distribution of DCpep-NM-PLGA-rOmpW (A). Zeta potential analyses of DCpep-NM-PLGA-rOmpW (B). SDS‒PAGE detection of surface proteins on DCpep-NM-PLGA C). \u0026nbsp;Ultraviolet‒visible near-infrared spectroscopy of FITC-DCpep, NM-PLGA and FITC-DCpep-NM-PLGA (D) TEM images of PLGA, NM, NM-PLGA and DCpep-NM-PLGA (E). Colocalization of PLGA, NM-PLGA and DCpep-NM-PLGA in mouse macrophages (F).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5642310/v1/1c8e03625e62c7d0320f8bae.png"},{"id":71988058,"identity":"31af4a66-806e-4d71-ae64-a23c061146da","added_by":"auto","created_at":"2024-12-20 11:09:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":197626,"visible":true,"origin":"","legend":"\u003cp\u003eStandard curve of the rOmpW concentration(A) and release curves of rOmpW in DCpep-NM-PLGA-rOmpW.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5642310/v1/27fd336b0d4e375550b8d148.png"},{"id":71985785,"identity":"5d8437e4-d358-4a1b-a320-22251a6ee765","added_by":"auto","created_at":"2024-12-20 10:45:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":386154,"visible":true,"origin":"","legend":"\u003cp\u003eImmune responses in BALB/c mice one week after the last immunization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes: \u003c/strong\u003eExpression levels of specific IgG (A), IgG1 (B) and IgG2a (C) in mouse serum. Expression levels of IFN-γ (D), IL-4 (E) and IL-17A (F) in splenocyte supernatants. The data are presented as the means ± SDs (n=3). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5642310/v1/c7427443a55f668575d6cd46.png"},{"id":71985777,"identity":"3f236fa6-92a6-478a-be66-778d2b680dba","added_by":"auto","created_at":"2024-12-20 10:45:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2163550,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry analysis of the draining lymph nodes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes: \u003c/strong\u003eFlow cytometry scatter plots of CD19\u003csup\u003e+\u003c/sup\u003e B cells, CD3\u003csup\u003e+\u003c/sup\u003e T cells(A), CD11c\u003csup\u003e+\u003c/sup\u003e DCs and F4/80\u003csup\u003e+\u003c/sup\u003e(B) macrophages in the lymph nodes 7 days after the last immunization. Statistical graphs of CD19\u003csup\u003e+\u003c/sup\u003e B cells(C), CD3\u003csup\u003e+\u003c/sup\u003e T cells(D), CD11c\u003csup\u003e+\u003c/sup\u003e DCs(E) and F4/80\u003csup\u003e+\u003c/sup\u003e macrophages(F).The data are presented as the means ± SDs (n=3). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5642310/v1/e661b38e34d066062de9e827.png"},{"id":71986628,"identity":"9fd075ef-3748-4e97-80fa-b68e1dd0c831","added_by":"auto","created_at":"2024-12-20 10:53:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1931294,"visible":true,"origin":"","legend":"\u003cp\u003eImmunoprotective effects of DCpep-NM-PLGA-rOmpW in BALB/c mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes: \u003c/strong\u003eBacterial loads in the blood (A) and lungs (B) of the mice at 24 hours postchallenge. Survival rates (C), body weight changes (D) and clinical scores (E) each day after \u003cem\u003eA. baumannii\u003c/em\u003echallenge. Lung tissue sections were stained with haematoxylin‒eosin and observed under a microscope (200×) (F). The data are presented as the means ± SDs (n=6). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5642310/v1/c4915a83fb73f0d2c6b853e6.png"},{"id":71986592,"identity":"914d0b78-5aa7-4aef-9c90-83ed8200280b","added_by":"auto","created_at":"2024-12-20 10:53:43","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":143282,"visible":true,"origin":"","legend":"\u003cp\u003eDuration of specific IgG in mouse serum. The data are presented as the means ± SDs (n=6). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5642310/v1/4dc629192ef3f7995adf168b.png"},{"id":72030305,"identity":"644cc21a-1791-40d5-9327-175473a02ccd","added_by":"auto","created_at":"2024-12-20 20:31:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16074236,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5642310/v1/5a86c690-fea5-44d7-9ccb-f8544ba54fd9.pdf"},{"id":71985767,"identity":"0d216c80-aa6e-4c8e-b02d-396eb4e1f521","added_by":"auto","created_at":"2024-12-20 10:45:43","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3872063,"visible":true,"origin":"","legend":"","description":"","filename":"file.docx","url":"https://assets-eu.researchsquare.com/files/rs-5642310/v1/42aeb2449c532b3238e4b0d9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Efficient Immune Shield: Biomimetic Nanovaccine of Acinetobacter Baumannii Recombinant Outer Membrane Protein W","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e (\u003cem\u003eA. baumannii\u003c/em\u003e) is a gram-negative opportunistic pathogen and a common pathogen of hospital-acquired infections. The widespread emergence of multidrug-resistant (MDR) and pandrug-resistant (PDR) strains poses a significant threat to human health\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Many scholars have been committed to developing new antibacterial drugs against \u003cem\u003eA. baumannii\u003c/em\u003e infections, but in the past 30 years, very few new antibiotics have been developed\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Therefore, finding new treatment methods other than new antibacterial drugs is urgent.\u003c/p\u003e \u003cp\u003eVaccines have been proven safe and effective in preventing many bacterial infections\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. However, none of them have yet to be applied in the clinic. Although the initial vaccines developed from whole-inactivated or live-attenuated strains were easy to prepare and had partial immune protection effects, the use of formaldehyde as the primary inactivating agent cannot fully inactivate \u003cem\u003eA. baumannii\u003c/em\u003e and poses potential safety concerns\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Some researchers have shown that the outer membrane of \u003cem\u003eA. baumannii\u003c/em\u003e contains more antigenic epitopes, such as outer membrane proteins and capsular polysaccharides, but only a few antigens play immune-protective roles\u003csup\u003e[\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. These proteins are complicated to extract, have lower purity, and are unsuitable for mass production.\u003c/p\u003e \u003cp\u003eOmpW, a highly conserved small molecule hydrophobic outer membrane pore protein in \u003cem\u003eA. baumannii\u003c/em\u003e, is involved in the absorption of iron ions, and is related to resistance to carbapenem antibiotics\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Some studies have shown that OmpW has active and passive immunoprotective effects on \u003cem\u003eA. baumannii-\u003c/em\u003einfected mice, but it significantly inhibits the proliferation of 293T and A549 cells\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. To address this shortcoming, we used bioinformatics software and immunological methods to predict and screen for optimal T-cell and B-cell epitopes on the \u003cem\u003eA. baumannii\u003c/em\u003e OmpW protein in model animals. Finally, we constructed and chemically synthesized the multiepitope peptide rOmpW by concentrating the screened dominant B-cell epitopes and the dominant T-cell epitopes, which preserved the immunogenicity of the protein and avoided harmful effects.\u003c/p\u003e \u003cp\u003eGiven the protein\u0026rsquo;s tiny size, lack of tertiary structure, short half-life, and susceptibility to proteolytic enzyme degradation \u003cem\u003ein vivo\u003c/em\u003e and the low immunogenicity of the antigenic epitope peptide molecule, adjuvants are needed to enhance the immune response. PLGA is a macromolecular synthetic copolymer formed by the aggregation of glycolic acid and lactic acid, and its degradation products are water and carbon dioxide, which are nontoxic to humans. Due to its excellent biocompatibility, encapsulation, and film properties, it was approved by the United States Food and Drug Administration (FDA) for clinical application in 1989. PLGA is an ideal carrier for epitope vaccines because of its effectiveness in high tissue biodistribution, significant controlled release, and enhancement of antigen presentation and the immune response\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, the body frequently recognizes and eliminates traditional nanodelivery systems as \"nonself\" substances, which makes targeted delivery to the lesion challenging. In recent years, a new biomimetic technique for preparing nanoparticles has used cell membrane components on their surfaces\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Some researchers have used neutrophil membrane (NM) to create drug carriers and biomimetic drug delivery systems. Moreover, some studies have shown that neutrophil membrane-encapsulated nanoparticle-mediated gentamicin can effectively resist drug-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e infection by preventing immune system clearance and achieving efficient targeted aggregation\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Dendritic cells (DCs) are specialized antigen-presenting cells that play essential roles in innate and acquired immune responses. DCpep is a targeting peptide with 12 amino acids (FYPSYHSTPQRP) that binds to receptors expressed on DCs. It participates in the endocytosis process, promoting immunogens to enter the cell more efficiently without impairing their function\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we screened out the dominant B-cell and T-cell antigenic epitopes, encapsulated them with PLGA and NM, surface-modified DCpep, and constructed the \u003cem\u003eA. baumannii\u003c/em\u003e biomimetic nanovaccine DCpep-NM-PLGA-rOmpW. Next, we investigated the immune efficacy of the biomimetic nanovaccine in model mice and observed its immunoprotective effect on \u003cem\u003eA. baumannii\u003c/em\u003e acute lethal pneumonia models to provide a laboratory basis for the clinical application of the biomimetic nanovaccine and potential candidate strategy for the prevention of \u003cem\u003eA. baumannii\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe ATCC17978 strain and the human lung adenocarcinoma cell strain A549 were maintained in our laboratory. PLGA (lactide: glycolide\u0026thinsp;=\u0026thinsp;50:50; MW\u0026thinsp;=\u0026thinsp;30,000\u0026ndash;60,000), IL-4, IFN-γ, IL-17A, IgG1, and IgG2a ELISA kits; human neutrophil extraction kits; and anti-mouse CD3, IgG, CD19, CD80, CD11c, F4/80, Taq enzyme, dNTP, T4 DNA ligase, BamH Ⅰ, Hind III, Nde I, Hind III and DNA marker antibodies were all purchased from Sigma‒Aldrich (St. Louis, MO).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eFemale BALB/c mice, aged 6\u0026ndash;8 weeks, were purchased from Shanghai Super-B\u0026amp;K Laboratory Animal Corp. Ltd. (Shanghai, China) and raised at the Laboratory Animal Center of Nanjing Medical University. All the animal experiments were approved by the Ethics Committee of Nanjing Medical University (Approval No. IACUC-2404066).\u003c/p\u003e\n\u003ch3\u003eExpression and Purification of OmpW\u003c/h3\u003e\n\u003cp\u003eSpecific PCR primers were designed according to the gene sequence encoding the OmpW protein: upstream primer: 5'-AAGGATCCGGGTAATTGGCAAGTAAAATTTG-3', downstream primer: 5'-TACTCGAGTTAGAATTTAGCTATAACC-3'. The following PCR conditions were used: thirty cycles of predenaturation (one minute at 98\u0026deg;C), denaturation (fifteen seconds at 98\u0026deg;C), annealing (thirty seconds at 58\u0026deg;C), and extension (thirty seconds at 72\u0026deg;C), followed by extension (one minute at 72\u0026deg;C) and storage (at 10\u0026deg;C). The target gene and empty plasmid were double digested using the restriction endonucleases BamH I and Xho I and then ligated and transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) competent cells. Then, the colonies were picked, the plasmids were extracted, and the target gene was enzymatically digested and identified. IPTG-induced recombinant and target protein expressions were analyzed by 15% SDS‒PAGE. The target recombinant proteins were purified using Ni-NTA affinity chromatography, and the OmpW protein concentration was determined using a bicinchoninic acid (BCA) protein quantification kit after endotoxin was removed from the recombinant protein mixture.\u003c/p\u003e\n\u003ch3\u003ePrediction and Identification of B-Cell Epitopes and T-Cell Epitopes of OmpW\u003c/h3\u003e\n\u003cp\u003e The B-cell epitopes of the OmpW protein were predicted using the OptimumAntigen Design Tool (GenScript, China) according to their secondary structure, surface accessibility, flexibility, hydrophilicity, and antigenic index. After comprehensive analysis, B-cell candidate epitope peptides with a length of 14 amino acids and no overlap were selected. (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). When predicting T-cell epitopes, the Immune Epitope Database Analysis Resource (IEDB) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tools.iedb.org/mhcii/\u003c/span\u003e\u003cspan address=\"http://tools.iedb.org/mhcii/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze and predict the A and E subregions of MHC-II genes in mice. (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). T-cell candidate epitopes with a length of 15 amino acids, no overlaps, and higher scores were stored. The predicted B-cell and T-cell epitope peptides were chemically synthesized by GenScript Biotechnology Co. Ltd (China).\u003c/p\u003e \u003cp\u003eTwelve BALB/c mice were randomly and equally divided into the OmpW group and the control group. The mice in the OmpW group were subcutaneously injected with 100 \u0026micro;g of whole-length OmpW protein mixed thoroughly with an equal volume of Freund's adjuvant, and the final volume was 100 \u0026micro;l. The mice in the control group were injected with an equal volume of PBS. Both groups were subcutaneously immunized three times at two-week intervals; one week after the final immunization, the specific IgG antibody levels in the serum were detected by indirect ELISA. In addition, spleen cells were isolated, adjusted to a concentration of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/ml, and stimulated with 20 \u0026micro;g/ml T-cell antigen epitopes. After 72 h, the supernatant was collected, and the gamma interferon (IFN-γ) level in the supernatant was measured using ELISA. Through analysis, we screened two dominant B-cell antigenic epitopes (amino acid sequences: EQGVADKVKEDFG and AKYHFKNSTRFTPY) and one dominant T-cell epitope (amino acid sequence: PSEDTTTALGVVKAD) of the OmpW protein and constructed the multiepitope peptide rOmpW.\u003c/p\u003e\n\u003ch3\u003eConstructed and Chemically Synthesized rOmpW\u003c/h3\u003e\n\u003cp\u003eThe peptides of two dominant B-cell epitopes (OmpW B1 and B6) and one T-cell epitope (OmpW T4) screened were connected in series by 6-aminohexanoic acid to construct rOmpW with 45 amino acids, synthesized by Gill Biochemistry Co. Ltd. (Shanghai). The molecular weight of rOmpW was 4974.60 Da as determined by mass spectrometry, and the purity of rOmpW was 95.50% as determined by high-performance liquid chromatography (HPLC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSafety of rOmpW\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEach 10 \u0026micro;l culture of OmpW (10, 20, 40, or 80 \u0026micro;g/ml) or rOmpW (10, 20, 40, or 80 \u0026micro;g/ml) was incubated with A549 cells for 24 or 48 h, respectively, and the level of A549 cell proliferation was detected by CCK-8 assay.\u003c/p\u003e \u003cp\u003e \u003cb\u003eValidity Assessment of rOmpW\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSixty BALB/c mice were divided randomly and equally into five groups: the OmpW, rOmpW (25, 50 and 100 \u0026micro;g), and control groups. 50 \u0026micro;g of whole-length OmpW protein was used in the OmpW group; and 25, 50, and 100 \u0026micro;g rOmpW mixed respectively and thoroughly with an equal volume of Freund's Adjuvant were utilized in the rOmpW groups with a final volume of 100 \u0026micro;l; the control group was given an equal volume of PBS. The mice were subcutaneously injected three times every two weeks. One week after the final immunization, the level of rOmpW specific-IgG antibody in the peripheral blood of the mice was detected by indirect ELISA, and the level of INF-γ secretion in the supernatant of the spleen cells was detected by ELISA.\u003c/p\u003e \u003cp\u003eTwo weeks after the final immunization, a lethal dose of the ATCC17978 (2\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU) strain was intratracheally administered to each mouse to establish acute lethal pneumonitis models of \u003cem\u003eA. baumannii\u003c/em\u003e. After 24 h, the peripheral blood and right lung tissue homogenate were collected for colony culture and counting. The left lung tissue was stained with HE to observe the pathological changes for seven consecutive days, and the survival status, weight changes, and symptom scores of the mice were recorded daily. The symptom scoring of the mice was mainly based on activity status, stooping, shut eyes, fur condition, and movement. Individual symptom scores were assigned as 0 (normal and active), \u0026minus;\u0026thinsp;1 (partially disorganized fur), \u0026minus;\u0026thinsp;2 (obviously disorganized fur, slight stooping), \u0026minus;\u0026thinsp;3 (a large amount of fur loss, obvious stooping, slow movement, shut eyes), \u0026minus;\u0026thinsp;4 (inability to stand or move), or \u0026minus;\u0026thinsp;5 (death).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Human Neutrophil Membranes\u003c/h2\u003e \u003cp\u003eBlood samples from 10 healthy adults (15 ml/person, 150 ml total) were collected using anticoagulant tubes (approved by the Second Affiliated Hospital of Nanjing Medical University, Approval No. 2023-KY-165-01). Neutrophils were extracted using a peripheral blood neutrophil isolation kit. A Diff-Ouik Stain kit was used to identify the neutrophils.\u003c/p\u003e \u003cp\u003eThe washed neutrophils were resuspended in hypotonic solution (PBS: ultrapure water\u0026thinsp;=\u0026thinsp;1:10) and lysed at room temperature for one hour. The lysed cells were subsequently pulverized in an ultrasonic cleaning tank at 4\u0026deg;C for 5 minutes and processed by middle-frequency ultrasonication. The first centrifugation was performed at 3500 rpm/4\u0026deg;C for five minutes, then a second centrifugation was performed at 4\u0026deg;C/14,500 rpm for 40 minutes. To obtain uniformly sized neutrophil membranes, we used a polycarbonate membrane with a pore size of 200 nm and extruded the neutrophil membranes with a miniextruder.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of DC-targeted peptides (DCpep)\u003c/h3\u003e\n\u003cp\u003eNanjing Putao Biotechnology Co. Ltd. was commissioned to chemically synthesize DCpep with 12 amino acids. The molecular weight of DCpep was identified as 1479.63 Da by mass spectrometry, and the purity of the synthesized peptide was 98.23% as determined by HPLC. To modify the cytomembrane using the phospholipid insertion method, we commissioned this Nanjing Putao Biotechnology to prepare DSPE-PEG2000-DCpep.\u003c/p\u003e\n\u003ch3\u003eConstruction of the Biomimetic Nanovaccine\u003c/h3\u003e\n\u003cp\u003erOmpW encapsulated with PLGA was prepared using a water/oil/water double emulsion evaporation technique. PLGA-rOmpW was mixed with human neutrophil membranes at a ratio of 5:1, after which the mixture was extruded using a miniextruder through a polycarbonate membrane with a pore size of 200 nm. The mixture was centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the mixture was resuspended in PBS to obtain NM-PLGA-rOmpW. DSPE-PEG2000-DCpep was added to the NM-PLGA-rOmpW solution at a ratio of 1:5. The mixture was stirred for two hours in the dark and then centrifuged at 10,000 rpm for 15 min. After that, the supernatant was discarded, and the precipitate was collected. Finally, the \u003cem\u003eA. baumannii\u003c/em\u003e biomimetic nanovaccine DCpep-NM-PLGA-rOmpW was obtained by resuspension in PBS buffer.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eParticle size, PDI, and Zeta Potential Measurement\u003c/h2\u003e \u003cp\u003eEach 3 ml emulsion of PLGA-rOmpW and DCpep-NM-PLGA-rOmpW was diluted with ultrapure water in moderation and placed in a cuvette. The average particle size, polydispersity index (PDI), and zeta potential were measured by a particle size analyser. Each sample was measured five times, and the average value was taken to ensure the accuracy of the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMembrane protein properties of DCpep-NM-PLGA\u003c/h2\u003e \u003cp\u003eWe performed polyacrylamide gel electrophoresis (SDS-PAGE) to validate the presence of neutrophil membrane proteins on the surface of DCpep-NM-PLGA. Each 20 \u0026micro;l solution of PLGA, NM, NM-PLGA, or DCpep-NM-PLGA was added to four electrophoretic channels. After the electrophoresis was completed, Coomassie Brilliant Blue solution was added to stain the gels for 30 minutes, followed by one hour of washing. The gels were then photographed and the images were recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTransmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eTo observe the morphology of the neutrophil membranes, PLGA, NM-PLGA and DCpep-NM-PLGA, the four substances were diluted 20 times with ultrapure water. A small amount of each dilution liquid was subsequently dropped on a copper grid and left for 2 minutes to allow the liquid to permeate naturally. Next, the excess liquid was gently removed from the surface of the copper grid with filter paper to ensure that it was dried naturally at room temperature. After drying, negative staining was performed with 0.1% phosphotungstic acid for 5 min, and the samples were subjected to transmission electron microscopy (TEM) to observe the microscopic morphology in detail.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eColocalization in cells\u003c/h2\u003e \u003cp\u003eWe commissioned China Pharmaceutical University to use PLGA to encapsulate Cyanine-3 (CY3), which is entirely insoluble in water and soluble in chloroform. The neutrophil membranes were incubated with DIL for 20 min in the dark and then centrifuged at 1500 rpm for 5 min, after which the precipitate was collected and resuspended in PBS. We mixed 0.5 mg of PLGA with 0.1 mg of neutrophil membranes, selected a PVDF membrane with a pore size of 200 nm, and physically extruded the mixture using a small mini extruder. Finally, we obtained the NM-PLGA reference for the phospholipid insertion method. FITC-DSPE-PEG2000-DCpep (0.1 mg) and 0.5 mg of NM-PLGA were stirred away from light for one hour and then centrifuged at 10,000 rpm for 5 min, after which the precipitate was removed and resuspended in PBS to obtain DCpep-NM-PLGA. Each 50 \u0026micro;l solution of PLGA, NM-PLGA, or DCpep-NM-PLGA was coincubated with RAW264.7 cells for one hour. The colocalization phenomenon in cells was observed using laser scanning confocal microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDetection of the Encapsulation Efficiency\u003c/h2\u003e \u003cp\u003eA standard curve was generated to measure the encapsulation rate of rOmpW in the \u003cem\u003eA. baumannii\u003c/em\u003e biomimetic nanovaccine, and the amount of unencapsulated rOmpW was indirectly calculated by HPLC. Finally, the encapsulation rate (EE) was calculated. Encapsulation rate (%) = (total rOmpW input - rOmpW amount in the supernatant)/total rOmpW input \u0026times; 100.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ePeptide Release\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFive milliliters of DCpep-NM-PLGA-rOmpW was placed in a shaker at 37\u0026deg;C and 100 rpm to simulate the release process under physiological conditions. The release of the rOmpW peptide was determined at various time intervals (1, 2, 4, 8, 13, 24, 30, 36, 48, 54, 60, and 72 h). After the samples were centrifuged at high speed, the supernatants were collected, and the rOmpW peptide concentration was accurately determined by HPLC.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSafety\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA549 cells were coincubated with 10 \u0026micro;L of PBS (as the control group) or different concentrations of DCpep-NM-PLGA-rOmpW (10, 20, 40, or 80 \u0026micro;g/ml rOmpW) for 24 or 48 h. The effects of these combinations on the proliferation of A549 cells were detected by CCK-8 assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of the grouping, immunization and modeling of mice\u003c/h2\u003e \u003cp\u003eSixty BALB/c mice were randomly and equally divided into four groups: the DCpep-NM-PLGA-rOmpW, DCpep-NM-PLGA-PBS, rOmpW and NS groups. Each group was immunized with 200 \u0026micro;l of DCpep-NM-PLGA-rOmpW containing 50 \u0026micro;g of rOmpW, 200 \u0026micro;l of DCpep-NM-PLGA-PBS, 50 \u0026micro;g of rOmpW dissolved in 100 \u0026micro;l of NS with 100 \u0026micro;l of Freund\u0026rsquo;s adjuvant, and 200 \u0026micro;l of saline. All the mice were subcutaneously immunized every two weeks for a total of 3 times. One week after the last immunization, the rOmpW-specific antibody level in the peripheral blood of the mice was detected by indirect ELISA, and the secretion levels of IgG1 and IgG2a in the serum and IL-4, IL-17A, and INF-γ in the supernatant of the spleen cells were detected by ELISA. Flow cytometry was used to detect the frequencies of CD19\u003csup\u003e+\u003c/sup\u003e, CD3\u003csup\u003e+\u003c/sup\u003e, CD11c\u003csup\u003e+\u003c/sup\u003e, and F4/80\u003csup\u003e+\u003c/sup\u003e cells in the inguinal lymph nodes.\u003c/p\u003e \u003cp\u003eTwo weeks after the final immunization, a lethal dose of ATCC17978 was administered via the trachea to establish the acute fatal pneumonia model of \u003cem\u003eA. baumannii\u003c/em\u003e. Colony counts in peripheral blood and lung tissue were performed 24 hours later. HE staining was used to observe pathological changes in the left lung tissue. The remaining mice were monitored for one week, and their survival status, weight changes, and clinical symptom scores were recorded daily.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSpecific Antibody Detection of rOmpW\u003c/h2\u003e \u003cp\u003eThe rOmpW was first diluted to 10 \u0026micro;g/mL to coat a 96-well plate. Then, the resulting mixture was added to each well (100 \u0026micro;L per well) and incubated at 4\u0026deg;C for 12\u0026ndash;18 h. After washing five times with PBS and 0.05% Tween 20 (PBST), 200 \u0026micro;L of 2% bovine serum albumin (BSA) was added to each well and incubated at 37\u0026deg;C for 2 h. Then, the mouse serum was diluted with PBS at a ratio of 1:200, and 100 \u0026micro;L was added to each well of the OmpW-immunized and PBS groups. The plates were washed five times after incubation at 37\u0026deg;C for 2 h. After that, 100 \u0026micro;L of freshly diluted goat anti-mouse IgG (diluted 1:10000) and horseradish peroxidase (HRP) were added to each well, and the plates were incubated at 37\u0026deg;C for 1 h. The plates were then washed five times, and 100 \u0026micro;L of tetramethylbenzidine solution (TMB) substrate was added to each well in the dark for 20 min. Finally, 50 \u0026micro;L of termination solution was added to each well, and the data were read at an OD of 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCytokine Detection\u003c/h2\u003e \u003cp\u003eOne week after the final immunization, the peripheral blood of the mice was collected. The expression levels of IgG1 and IgG2a antibodies in the serum were detected using IgG1 and IgG2a ELASA kits. Splenocytes were isolated and adjusted to a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL. Two hundred microlitres of cell suspension was added to each well of a 96-well plate, the cells were stimulated with rOmpW (20 \u0026micro;g/mL), and the supernatant was collected after 72 h. The levels of IFN-γ, IL-4, and IL-17A were detected using IFN-γ, IL-4, and IL-17A ELISA kits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometric Analysis\u003c/h2\u003e \u003cp\u003eOne week after the final immunization, single-cell suspensions of inguinal lymph nodes were prepared. The cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/mL) were blocked with Fc blocking antibody for 15 min at 4\u0026deg;C. The cells were washed and stained with fluorescent-conjugated antibodies against lymphocyte surface receptors, APC-Mouse I-Ab, PerCP-Cy5.5-CD11c, PE-F4/80, PE-CD3 and APC-CD19 (BD Biosciences) at 4\u0026deg;C and protected from light for 30 min. Then, the cells were washed and fixed with 2% paraformaldehyde solution for 20 min. Data were acquired on a BD FACS Canto II flow cytometer (BD Biosciences) with at least 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e events for each sample and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHE staining of lung tissue\u003c/h2\u003e \u003cp\u003eTwenty-four hours after mice were challenged with \u003cem\u003eA. baumannii\u003c/em\u003e ATCC17978, the lungs were removed under aseptic conditions and fixed in 4% formalin. After being stained with haematoxylin‒eosin (HE), the paraffin-embedded sections were examined histopathologically by microscopy. Using a macro in ImagePro software, lung injury was estimated by the percentage of the lesion area among the total lung area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Statistical Package of Social Sciences (SPSS, version 25.0, SPSS Inc,Chicago,IL). T-test was chosen to compare the mean values between the two samples. One-way analysis of variance (ANOVA) was used to compare multiple variances and determine the homogeneity of variance analysis. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated that the difference was statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eExpression and Identification of the OmpW Protein\u003c/h2\u003e \u003cp\u003eThe recombinant plasmid pET28a-OmpW was verified by PCR amplification and digestion, and the results of the sequencing analysis are shown in Figure S2. The constructed recombinant plasmid pET-28a-OmpW was transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) BL21 cells, which were subsequently induced with IPTG. The OmpW protein was expressed in the form of an inclusion body, and the final concentration of the OmpW protein was 1.8 mg/mL, as measured by protein quantitative electrophoresis. The results of SDS‒PAGE revealed a specific protein band at 23 kDa, which was consistent with the expected size of the OmpW protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eEpitopes Prediction and Construction of the Multiepitope peptide rOmpW\u003c/h2\u003e \u003cp\u003eThrough bioinformatics analysis, six B-cell candidate epitope peptides (named OmpW B1\u0026ndash;B6) and four T-cell candidate epitope peptides (named OmpW T1\u0026ndash;T4) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were predicted and chemically synthesized.\u003c/p\u003e \u003cp\u003eBALB/c mice were subcutaneously injected with 100 \u0026micro;g of recombinant OmpW (1 \u0026micro;g/\u0026micro;L in PBS) or an equivalent volume of PBS three times every two weeks. The vaccination schedule is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The peripheral blood and spleen cells were collected one week after the final immunization. OmpW B1 epitopes, B6 epitopes and full-length proteins showed significant antigen-antibody reactions with the serum of the OmpW-immunized group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), and the T4 peptide significantly increased the level of IFN-γ secreted by the splenocytes of OmpW-immunized mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe screened peptides of two dominant B-cell epitopes (OmpW B1 and B6) and one T-cell epitope (OmpW T4) were connected in series using 6-aminohexanoic acid to design and generate a novel multiepitope peptide, rOmpW. rOmpW, with 45 amino acids and a molecular weight of 4974.60 Da, was identified by mass spectrometry (Figure S3A) and HPLC (Figure S3B).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eImmunoprotective Effects Of rOmpW\u003c/h2\u003e \u003cp\u003eBALB/c mice were subcutaneously injected with OmpW (50 \u0026micro;g), rOmpW (25, 50, or 100 \u0026micro;g), or an equal volume of PBS three times every two weeks. The peripheral blood and spleens were collected one week after the final immunization. The specific IgG antibody levels in the serum were detected by indirect ELISA. The OD450 nm values of the OmpW and rOmpW groups were significantly greater than those of the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the OD450 nm values of the 50 and 100 \u0026micro;g rOmpW groups were considerably greater than those of the OmpW and 25 \u0026micro;g rOmpW groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). IFN-γ in the supernatant secreted by splenocytes was detected by ELISA. The splenocytes from the OmpW and rOmpW groups produced higher levels of IFN-γ than those from the control group did (***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the level of IFN-γ in the 50 \u0026micro;g rOmpW group was significantly greater than that in the 25 \u0026micro;g rOmpW group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas there was no statistically significant difference from that in the 100 \u0026micro;g rOmpW group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe evaluation of the lethal dose of \u003cem\u003eA. baumannii\u003c/em\u003e ATCC17978 is shown in Figure S4. When 100 \u0026micro;l of \u003cem\u003eA. baumannii\u003c/em\u003e ATCC17978 (2\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/ml) was injected into the trachea, the 3-day survival rate was 0%. For the subsequent experiments, we selected 100 \u0026micro;l of \u003cem\u003eA. baumannii\u003c/em\u003e ATCC17978 (2\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/ml) administered via intratracheal injection to establish an acute fatal pneumonia model. To determine whether rOmp22 conferred protection, we assessed its effectiveness in an acute pneumonia model in BALB/c mice. Two weeks after the last boost immunization, all the groups were challenged with lethal doses of \u003cem\u003eA. baumannii\u003c/em\u003e ATCC17978 (2\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/ml). The number of CFUs was counted in the peripheral blood and tissue from the right lung at 24 h post challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). Compared with control mice, OmpW and rOmpW immunized mice presented lower bacterial loads in the blood and lung tissue (***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The colony counts of the blood and lung tissue in the rOmpW 50 \u0026micro;g group were lower than those in the OmpW and rOmpW 25 \u0026micro;g groups (**P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and there was no statistically significant difference from the rOmpW 100 \u0026micro;g group. After 24 hours, the left lung tissue was stained with HE and analysed semiquantitatively using Image-Pro Plus software (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), and the areas of inflammation in the OmpW and rOmpW groups were significantly lower than that in the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In addition, the area of inflammation in the 50 \u0026micro;g rOmpW group was smaller than that in the 25 \u0026micro;g rOmpW group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, there was no statistically significant difference in the rOmpW 100 \u0026micro;g group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eSix mice challenged with \u003cem\u003eA. baumannii\u003c/em\u003e were randomly selected for recording survival, body weight changes and clinical scores every day for seven days (Figure S5A‒C). The survival rates in the rOmpW 25 \u0026micro;g, rOmpW 50 \u0026micro;g, rOmpW 100 \u0026micro;g, and OmpW groups were 50% (3/6), 66.67% (4/6), and 66.67% (4/6), respectively. A survival rate of 50% (3/6) was significantly greater than that in the control group (3-day survival rate of 0). The body weights of the mice in each group decreased to the lowest levels on approximately the second day; after that, the body weights of the surviving mice in the OmpW and rOmpW groups gradually increased and then recovered to the level observed before the challenge on the 6th to 7th days. The clinical scores of the mice reached the lowest level on the second day after the challenge, and the mice in the control group died within three days. The clinical scores of the surviving mice in the OmpW and rOmpW groups gradually increased, and the symptom scores of the surviving mice generally returned to 0 points on the sixth day of observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eEffects of rOmpW on the Proliferation Level of A549 cells\u003c/h2\u003e \u003cp\u003eWe used the CCK-8 method to detect the proliferation level of A549 cells to evaluate the \u003cem\u003ein vitro\u003c/em\u003e safety of rOmpW (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Different concentrations of rOmpW were cocultivated with A549 cells for 24 and 48 hours, and there was no significant inhibitory effect on the growth of A549 cells, whereas OmpW significantly inhibited the growth of A549 cells at a concentration of 20 \u0026micro;g/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003ePreparation of Neutrophil Membranes and DCpep\u003c/h2\u003e \u003cp\u003ePeripheral blood neutrophils from healthy adults were extracted using a Peripheral Blood Neutrophil Isolate Kit and identified using a CDiff-Quik Stain Kit (Figure S6). Neutrophil membranes were obtained by ultracentrifugation. DCpep, with 12 amino acids and a molecular weight of 1479.63 Da, was chemically synthesized and identified by mass spectrometry (Figure S7A) and HPLC (Figure S7B).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization and Identification of Biomimetic Nanoparticles\u003c/h2\u003e \u003cp\u003eThe average particle size of DCpep-NM-PLGA-rOmpW was 135.67\u0026thinsp;\u0026plusmn;\u0026thinsp;11.26 nm(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), the PDI was 28.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.50%, and the zeta potential was \u0026minus;\u0026thinsp;10.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). SDS‒PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) revealed that DCpep-NM-PLGA, NM-PLGA, and NM exhibited similar electrophoretic bands, indicating that DCpep-NM-PLGA prepared by extrusion could better maintain the integrity of the protein on the surface of the neutrophil membrane. We modified DCpep with FITC, and FITC-DCpep resulted in an obvious absorption peak at 495 nm in the UV\u0026ndash;vis spectrum. The aqueous solution of FITC-DCpep-NM-PLGA also showed an obvious absorption peak at 495 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), which indicated that DCpep could be successfully modified on the surface of human neutrophil membranes. Transmission electron microscopy (TEM) was performed to assess the morphology and size of the biomimetic nanoparticles. PLGA, NM, and DCpep-NM-PLGA were analyzed by TEM, and the results revealed that the neutrophil membranes could be better attached to the surface of PLGA without affecting the size of the particles and that DCpep-NM-PLGA exhibited spherical structures that were uniform and well dispersed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Fifty microlitres of PLGA, NM-PLGA, or FITC-DCpep-NM-PLGA was cocultured with RAW264.7 cells. After 1 h of incubation, the samples were observed by laser scanning confocal microscopy. Significant colocalization phenomena among DCpep, neutrophil membranes and PLGA could be clearly observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEncapsulation Efficiency and Release Studies\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe standard curve was constructed by HPLC with the rOmpW concentration as the horizontal coordinate and the peak area at the corresponding concentration as the vertical coordinate. The equation of this standard curve was y\u0026thinsp;=\u0026thinsp;11385x\u0026thinsp;\u0026minus;\u0026thinsp;217.23, and the R\u0026sup2; value was 0.9998, indicating a good linear relationship (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The encapsulation efficiency (EE) of DCpep-NM-PLGA-rOmpW was indirectly calculated to be 43.25% by HPLC, which is in line with expectations. DCpep-NM-PLGA-rOmpW was dissolved in 5 ml of physiological saline solution (pH 7.4) in a shaker at 37\u0026deg;C and 100 rpm. The rOmpW content in the supernatant was determined by HPLC. The cumulative release rates of rOmpW from DCpep-NM-PLGA-rOmpW at 1, 2, 4, 8, 13, 24, 48 and 72 h were 17.7%, 23.3%, 27.7%, 32.7%, 33.8%, 34.3%, 38.8% and 44.3%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), revealing a significant burst release of rOmpW at 1 h, followed by a gradual and slow release that could be sustained for 72 h, indicating that the biomimetic nanovaccine had a longer protection time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eImmunization Efficacy of the Biomimetic Nanovaccine\u003c/h2\u003e \u003cp\u003eBALB/c mice were immunized subcutaneously with DCpep-NM-PLGA-rOmpW containing 50 \u0026micro;g/200 \u0026micro;l of rOmpW, an equal volume of DCpep-NM-PLGA-PBS, 50 \u0026micro;g of rOmpW or an equal volume of saline every two weeks for a total of three immunizations. The peripheral blood and spleens were collected one week after the last vaccination. The levels of rOmpW-specific IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), IgG1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), and IgG2a (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) in the serum were detected by ELISA. Compared with those in the NS, rOmpW, and DCpep-NM-PLGA-PBS groups, the expression levels of specific IgG, IgG1, and IgG2 antibodies in the serum of the mice in the biomimetic nanovaccine group were significantly greater, suggesting that the biomimetic nanovaccine could induce a stronger humoral immune response. The secretion levels of IFN-γ and IL-17A in the \u003cem\u003eA. baumannii\u003c/em\u003e nanovaccine group were also significantly greater (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) than those in the NS, rOmpW, and DCpep-NM-PLGA-PBS groups, and the expression levels of IFN-γ, IL-4, and IL-17A in the supernatants of the spleen cells were detected by ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F). However, the expression level of IL-4 in spleen cells was significantly decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting that the biomimetic nanovaccine induced stronger Th1 and Th17 cellular immune responses.\u003c/p\u003e \u003cp\u003eOne week after the final immunization, flow cytometry was used to detect the percentages of CD19\u003csup\u003e+\u003c/sup\u003e, CD3\u003csup\u003e+\u003c/sup\u003e, CD11c\u003csup\u003e+\u003c/sup\u003e, and F4/80\u003csup\u003e+\u003c/sup\u003e cells in the single-cell suspensions of the inguinal lymph nodes in each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-F). The frequencies of CD3\u003csup\u003e+\u003c/sup\u003e T lymphocytes, CD19\u003csup\u003e+\u003c/sup\u003e B lymphocytes, CD11c\u003csup\u003e+\u003c/sup\u003e DCs, and F4/80\u003csup\u003e+\u003c/sup\u003e macrophages in the biomimetic nanovaccine group were significantly greater than those in the NS, rOmpW and DCpep-NM-PLGA-PBS groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings indicated that the biomimetic nanovaccine could stimulate the differentiation of B and T lymphocytes in the lymph nodes and promote the migration of DCs and macrophages to the lymph nodes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003eImmunoprotective Effects of Biomimetic Nanovaccine\u003c/b\u003e\u003c/div\u003e \u003cp\u003eTo determine whether the biomimetic nanovaccine conferred immune protection, we assessed its effectiveness in an acute pneumonia model in BALB/c mice. All the groups were challenged with lethal doses of ATCC17978 via the trachea. The bacterial loads in the peripheral blood and lung tissue of the mice immunized with the biomimetic nanovaccine were lower than those in the peripheral blood and lung tissue of the mice in the other three groups 24 h postchallenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003eThe survival status, weight changes, and clinical scores of the mice in each group were recorded for seven consecutive days after the \u003cem\u003eA. baumannii\u003c/em\u003e ATCC17978 challenge. The one-week survival rates were 83.33% (5/6) in the biomimetic nanovaccine group and 66.67% (4/6) in the rOmpW group, whereas all the mice in the NS and DCpep-NM-PLGA-PBS groups died within three days (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). The weight of the mice in each group decreased to the lowest level on approximately the second day after \u003cem\u003eA. baumannii\u003c/em\u003e challenge, and the weights of the surviving mice in the biomimetic nanovaccine and rOmpW groups gradually increased and generally recovered to the preattack levels on approximately the seventh day (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Symptom scores decreased to the lowest level in each group on the 2\u0026ndash;3 days after \u003cem\u003eA. baumannii\u003c/em\u003e challenge. The symptom scores of surviving mice in the biomimetic nanovaccine and the rOmpW groups gradually increased and generally returned to normal levels on the seventh day (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eHE staining was used to observe pathological changes in the left lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF), and the results revealed substantial inflammatory cell infiltration (mainly neutrophilic), necrosis of the lung tissue and damage to the structure of the alveolar lumen in the NS and DCpep-NM-PLGA-PBS groups. Moreover, the lung tissue in the rOmpW group was infiltrated by a few inflammatory cells, the interstitial lung tissue was thickened slightly, and the structure of the alveolar lumen was intact. However, in the biomimetic nanovaccine group, although a small number of inflammatory cells infiltrated the lung tissue, there was no edema or thickening in the interstitium of the lung tissue. The Image-Pro Plus software analysis results revealed that the inflammatory area of the lung tissue in the biomimetic nanovaccine group was significantly lower than those in the other three groups (each P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eDuration of rOmpW-Specific IgG antibodies in serum\u003c/h2\u003e \u003cp\u003eThe specific IgG level in the serum was detected by indirect ELISA at five time points: 1, 3, 5, 7, and 9 weeks after the final immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e), and venous blood was collected from the inner canthal orbital vein of each mouse. The OD value of the serum-specific IgG in the biomimetic nanovaccine group was significantly higher than those in the other three groups at all five time points (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In the ninth week, the serum-specific IgG level in the biomimetic nanovaccine group remained high. In contrast, the serum-specific IgG level in the rOmpW group gradually decreased in the 5th week and was not significantly different from that in the NS group at the 9th week (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eSafety of the Biomimetic Nanovaccine\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eToxicity is a primary concern when vaccines are used in biomedical applications. We tested the toxicity of the biomimetic nanovaccine in A549 human lung adenocarcinoma epithelial cells and in mice. The results showed that the biomimetic nanovaccine had no inhibitory effect on A549 cells (Figure S8A-B). One week after the final immunization, the serum and major organs of the mice were collected to detect changes in the serum levels of BUN, Cre, ALT, and AST. Histological changes in the heart, liver, spleen, lung, and kidney were assessed by HE staining (Figure S8C-G). The biomimetic nanovaccine caused no apparent damage to liver or kidney functions or the pathology of major organs (heart, liver, spleen, lungs, and kidneys). These results indicate that the biomimetic nanovaccine has a preeminent safety profile.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e is a strictly aerobic gram-negative bacillus that is highly resistant to humidity, ultraviolet radiation, and chemical disinfectants and can cause pneumonia, meningitis, urinary tract infections, bloodstream infections, and other disorders\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The resistance mechanism of \u003cem\u003eA. baumannii\u003c/em\u003e is complicated, and it is resistant to all commonly used clinical antibiotics. The development of new antimicrobial drugs against \u003cem\u003eA. baumannii\u003c/em\u003e takes a long time and also tends to induce new resistance in \u003cem\u003eA. baumannii\u003c/em\u003e. Vaccines are effective ways to prevent and control \u003cem\u003eA. baumannii\u003c/em\u003e infection. The development of vaccines against \u003cem\u003eA. baumannii\u003c/em\u003e has become a hot topic for many researchers\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOmpW is an important outer membrane protein of \u003cem\u003eA. baumannii\u003c/em\u003e. Hypoxia can lead to the downregulation of OmpW expression, resulting in decreased adhesion and invasion of \u003cem\u003eA. baumannii\u003c/em\u003e to human lung epithelial cells and reduced biofilm formation by \u003cem\u003eA. baumannii\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The OmpW protein is highly conserved among \u003cem\u003eA. baumannii\u003c/em\u003e strains. The homology of OmpW is more than 91% among 804 \u003cem\u003eA. baumannii\u003c/em\u003e strains. OmpW significantly reduces bacterial loads in various organs in \u003cem\u003eA. baumannii\u003c/em\u003e infection models and improves survival in mice. It also has passive immunoprotective benefits. However, OmpW has a slight inhibitory effect on the proliferation of 293T and A549 cells, suggesting that OmpW may have some toxicity\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In this study, the dominant antigenic epitopes of OmpW were predicted using bioinformatics methods and screened out by immunological means in vivo. The selected dominant antigenic epitopes were concatenated with 6-aminohexanoic acid and chemically synthesized to construct a multiepitope peptide, rOmpW. rOmpW was verified to have no inhibitory effect on the growth of A549 cells in vitro and to have an immunoprotective impact in vivo. The results showed that rOmpW induced stronger humoral and cellular immune responses in mice and increased their productivity. When the immunization dose of rOmpW was 50 \u0026micro;g, the levels of specific IgG in the serum and Th1 cytokines (IFN-γ) in the supernatant secreted by the spleen cells were significantly greater than those in the OmpW and rOmpW 25 \u0026micro;g groups, and there was no significant difference from those in the rOmpW 100 \u0026micro;g group. Therefore, 50 \u0026micro;g of rOmpW was considered an appropriate dose for a single immunization, which laid the foundation for subsequent experiments.\u003c/p\u003e \u003cp\u003eDue to the small size of the rOmpW segments, they are easily degraded by proteases in vivo, and their immunogenicity is relatively weak. To overcome these limitations and enhance the immunogenicity of rOmpW, we used PLGA as an adjuvant and carrier. However, conventional nano delivery systems are often cleared by the body as \"not self\", and the direct fusion of cell membranes with nanoparticles, a new method developed in recent years, reduces the clearance of the nanoparticles by the immune system. Neutrophil membranes have been proven to offer the advantages of immune escape and specific targeting in infections and tumors, among other effects\u003csup\u003e[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In this study, we extracted neutrophils from the peripheral blood of healthy adults and successfully prepared neutrophil membranes by ultracentrifugation. To reduce immune clearance, biomimetic modification of PLGA-rOmpW with neutrophil membranes was performed.\u003c/p\u003e \u003cp\u003eDue to their strong antigen-presenting ability, presenting dendritic cells (DCs) play a crucial role in natural and acquired immune responses to pathogens. The molecules targeted by DCs are essential in developing targeted drugs and vaccines for DCs. Dendritic cell-targeting peptide (DCpep) specifically binds to the receptors expressed on DCs, promoting the migration and activation of DCs, enhancing the delivery of immunogens to T and B lymphocytes, and activating Th1, Th2, and Th17 immune responses\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In this study, DCpep-modified NM-PLGA was used for the first time to prepare a biomimetic nanovaccine.\u003c/p\u003e \u003cp\u003eWe successfully prepared PLGA-rOmpW using a water/oil/water double emulsion evaporation technique and obtained NM-PLGA-rOmpW by physical extrusion. By applying the phospholipid insertion method, we successfully prepared DCpep-NM-PLGA-rOmpW by surface modification of DCpep\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. The characterization of DCpep-NM-PLGA-rOmpW revealed that the particle size was less than 200 nm, with a smooth spherical structure, uniform particle size, and good dispersion, as determined by transmission electron microscopy. In this study, we observed the release characteristics of DCpep-NM-PLGA-rOmpW in vitro, and the results revealed that rOmpW presented a biphasic pattern of initial burst release followed by sustained release \u003cem\u003ein vitro\u003c/em\u003e. Early rapid release can trigger a robust immune response. In contrast, the later sustained and slow release could continuously stimulate the body to produce antibodies and related cytokines, thus enhancing the immune response. Safety is the key to whether the nanovaccine can be used \u003cem\u003ein vivo\u003c/em\u003e. Therefore, we evaluated the safety of the biomimetic nanovaccine \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. We found that it had no inhibitory effect on the proliferation of A549 cells and did not damage liver or kidney functions or important organs, indicating that the biomimetic nanovaccine is safe and non-toxic and can be used for experimental studies \u003cem\u003ein vivo.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eTo verify the immunoprotective effect of the biomimetic nanovaccine, we examined the levels of the humoral immune response (rOmpW-specific IgG, IgG1, and IgG2a in serum) and the cellular immune response (IFN-γ, IL-4, and IL-17A in the supernatants of splenocytes) in mice. Humoral immunity is one of the most important mechanisms by which the body fights against pathogens, and the levels of specific antibodies in serum and their subtypes constitute an essential basis for evaluating the humoral immune response induced by vaccines\u003csup\u003e[\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. This study revealed that the biomimetic nanovaccine could increase the levels of specific IgG, IgG1 and IgG2a in the serum of mice. CD4\u003csup\u003e+\u003c/sup\u003e T cells can be differentiated into Th1, Th2 and Th17 subpopulations. Th1 cells mainly secrete cytokines such as IL-2 and IFN-γ to mediate the cellular immune response. Th17 cells secrete IL-17A, which can induce the production of many chemokines, facilitate the recruitment of neutrophils, and play a key role in bacterial clearance\u003csup\u003e[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. We found that the biomimetic nanovaccine could induce stronger Th1 (IFN-γ) and Th17 (IL-17A) immune responses. In addition, antibody duration is also an important indicator for evaluating vaccine effects\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. We found that the level of the rOmpW-specific IgG antibody induced by the biomimetic nanovaccine could be maintained more persistently, suggesting that the biomimetic nanovaccine may have a more prolonged immunoprotective effect. Moreover, flow cytometry revealed significant increases in the frequencies of CD11\u003csup\u003e+\u003c/sup\u003e DCs, F4/80\u003csup\u003e+\u003c/sup\u003e macrophages, CD3\u003csup\u003e+\u003c/sup\u003e T lymphocytes and CD19\u003csup\u003e+\u003c/sup\u003e B lymphocytes in the lymph nodes, suggesting that part of the mechanism of the immune response induced by the biomimetic nanovaccine may be related to promoting the migration of DCs and macrophages to the lymph nodes and stimulating the differentiation of B and T lymphocytes.\u003c/p\u003e \u003cp\u003eEstablishing an infection model is a key step in evaluating the protective effect of vaccines\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. We constructed an acute lethal pneumonia model of \u003cem\u003eA. baumannii\u003c/em\u003e infection by injecting a lethal dose of a standard \u003cem\u003eA. baumannii\u003c/em\u003e strain into the trachea in mice. We found that the biomimetic nanovaccine significantly reduced the bacterial load in the lung tissue and blood, improved the symptom scores, and reduced the mortality rate of the mice in the acute lethal pneumonia model. These findings indicate that the biomimetic nanovaccine DCpep-NM-PLGA-rOmpW can exert effective immunoprotection.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, a novel biomimetic nanovaccine, DCpep-NM-PLGA-rOmpW, was prepared. This biomimetic nanovaccine could promote the migration of DCs and macrophages to the lymph nodes, induce the proliferation of B and T lymphocytes, enhance the humoral immune response and Th1 and Th17 cellular immune response, and exert a better immunoprotective benefit. Therefore, this biomimetic nanovaccine is a desirable candidate vaccine for preventing \u003cem\u003eA. baumannii\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the contributions of Dr. Naveen Nagiah, a former postdoc at IMSTEL, for initiating this study. We also extend our appreciation to Dr. Sylvia L. Natividad and her student, Jose Perez, for their assistance in acquiring the videos. Furthermore, we acknowledge the support provided by the NIH-NIMHD-RCMI Grant no. 5G12MD007592, which facilitated the utilization of the confocal microscopy facility in BBRC at UTEP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIH and GC: Data curation, Formal Analysis, Investigation, Methodology, Project administration, Software, Visualization, Validation, Writing \u0026ndash; Original draft, Writing \u0026ndash; review \u0026amp; editing; AEP: Data curation, Formal analysis, Investigation, Software, Visualization, Writing \u0026ndash; review \u0026amp; editing. CDL: Conceptualization, Investigation, Methodology, Project administration, Writing \u0026ndash; review \u0026amp; editing; BJ: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Visualization, Resources, Supervision, Validation, Writing \u0026ndash; Original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from the National Institutes of Health (NIH 1SC1HL154511-01) and the National Science Foundation (NSF 1927628) awarded to BJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting Interests\u003c/em\u003e\u003c/strong\u003e: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported. However, it is important to note that BJ is a member of the editorial board of this journal. This relationship has been disclosed to ensure transparency in the review process.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDandachi I, Azar E, Hamouch R, Maliha P, Abdallah S, Kanaan E, Badawi R, Khairallah T, Matar GM, Daoud Z. Acinetobacter spp in a Third World Country with Socio-economic and Immigrants Challenges. J Infect Dev Ctries. 2019;13(11):948\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolmes AH, Moore LS, Sundsfjord A, Steinbakk M, Regmi S, Karkey A, Guerin PJ, Piddock LJ. Understanding the mechanisms and drivers of antimicrobial resistance. Lancet. 2016;387(10014):176\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu Y, Zhang X, Deng S, Yue C, Jia X, Lyu Y. 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Human dendritic cell targeting peptide can be targeted to porcine dendritic cells to improve antigen capture efficiency to stimulate stronger immune response. Front Immunol. 2022;13:950597.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu CM, Zhang L, Aryal S, Cheung C, Fang RH, Zhang L. Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform. Proc Natl Acad Sci U S A. 2011;108(27):10980\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaratovskaya A, Rudenko N, Zamyatina A, Zvonarev A, Oleinikov V, Shpirt A, Perepelov A, Knirel Y, Brovko F. Protective capacity of monoclonal antibodies against Acinetobacter baumannii K9 capsular polysaccharide. Microbiol Spectr. 2023;11(1):e0414122.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePulido MR, Garc\u0026iacute;a-Quintanilla M, Pach\u0026oacute;n J, McConnell MJ. A lipopolysaccharide-free outer membrane vesicle vaccine protects against Acinetobacter baumannii infection. Vaccine. 2020;38(4):719\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a-Quintanilla M, Pulido MR, Pach\u0026oacute;n J, McConnell MJ. Immunization with lipopolysaccharide-deficient whole cells provides protective immunity in an experimental mouse model of Acinetobacter baumannii infection. PLoS ONE. 2014;9(12):e114410.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan Z, Yang J, Hu R, Hu X, Chen K. Acinetobacter baumannii infection and IL-17 mediated immunity. Mediators Inflamm, 2016, 2016: 9834020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReidel IG, Curti CC, Dor\u0026eacute;mus L, B\u0026eacute;r\u0026eacute; E, Delwail A, Russi RC, Lecron JC, Morel F, Garc\u0026iacute;a MI, M\u0026uuml;ller DM, J\u0026eacute;gou JF, Veaute CM. 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J Infect. 2019;78(2):106\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eLocation and sequence of OmpW B-cell and T-cell antigen epitopes.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEpitopes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLocation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAmino acid sequence\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOmpW\u0026nbsp;B1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e124\u0026ndash;137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEQGVADKVKEDFGV\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOmpW\u0026nbsp;B2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u0026ndash;93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLLDGQKVARIKQLP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOmpW\u0026nbsp;B3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e161\u0026ndash;174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYADISPEVTLTNGA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOmpW\u0026nbsp;B4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u0026ndash;47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSEDTTTALGVVKAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOmpW B5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e147\u0026ndash;160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePADAKNWGVFVDVR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOmpW B6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98\u0026ndash;111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAKYHFKNSTRFTPY\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOmpW\u0026nbsp;T1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u0026ndash;34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGNWQVKFGGSVIAPS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOmpW\u0026nbsp;T2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u0026ndash;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADHEYAFTPSVEYFF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOmpW\u0026nbsp;T3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e105\u0026ndash;119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSTRFTPYIGIGATAF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOmpW\u0026nbsp;T4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33\u0026ndash;47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePSEDTTTALGVVKAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Acinetobacter baumannii, biomimetic nanovaccine, PLGA, neutrophil membrane, DCpep","lastPublishedDoi":"10.21203/rs.3.rs-5642310/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5642310/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eThe development of vaccines is an effective and safe strategy to combat multidrug-resistant (MDR) \u003cem\u003eAcinetobacter baumannii \u003c/em\u003e(\u003cem\u003eA. baumannii\u003c/em\u003e) infections. This study aimed to prepare a DC-targeting multiepitope peptide biomimetic nanovaccine and evaluate its immune response and protective effect in mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eThe B-cell and T-cell epitopes of the OmpW protein from \u003cem\u003eA. baumannii \u003c/em\u003ewere predicted and screened using bioinformatics methods and identified by immunological means. The selected dominant epitopes were conjugated in series with 6-aminocaproic acid, and a multiepitope peptide, rOmpW, was chemically synthesized. Then, rOmpW was encapsulated with polylactic-co-glycolic acid (PLGA) and a neutrophil membrane (NM), and the surface was modified with DC-targeting peptide (DCpep) to construct the biomimetic nanovaccine DCpep-NM-PLGA-rOmpW. This biomimetic nanovaccinecan induce strong Th1 and Th17 cellular immune responses and humoral immunity. The biomimetic nanovaccine produced efficient immunological protection in an acute lethal pneumonia model of \u003cem\u003eA. baumannii\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e Our results indicate the potential translational value of this biomimetic nanovaccinefor preventing \u003cem\u003eA.baumannii\u003c/em\u003e infection.\u003c/p\u003e","manuscriptTitle":"Efficient Immune Shield: Biomimetic Nanovaccine of Acinetobacter Baumannii Recombinant Outer Membrane Protein W","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-20 10:45:38","doi":"10.21203/rs.3.rs-5642310/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e1f6c781-15fd-46b0-ad34-42ac8e9a3532","owner":[],"postedDate":"December 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-20T20:23:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-20 10:45:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5642310","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5642310","identity":"rs-5642310","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}