Mice, strains and serum samples
Six- to eight-week-old specific pathogen-free female BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Company Limited (Beijing, China). PA XN-1 (CCTCC M2015730) was isolated from the sputum of a patient with severe pneumonia at Southwest Hospital in Chongqing, China . Sera were collected from PA-infected convalescent patients and healthy donors. Written informed consent forms (ICFs) were collected from all participants. All animal care and experiments complied with ethical regulations and were approved by the Animal Ethical and Experimental Committee of the Third Military Medical University (No. AMUWEC2020967).
Screening immune-dominant epitopes
After evaluation of the transcriptome results, eight transmembrane proteins with the most significant changes in mRNA levels were selected. The protein information is listed in Additional file 1: Table S1. The structures of PA1777, PA4067, PA0595, PA0958 and PA2398 are from the PDB database, while the structures of PA1178, PA4554 and PA0165 were modeled by SWISS-MODEL and validated by Procheck and QMEAN [19, 20]. Then, PRED-TMBB (http://bioinformatics.biol.uoa.gr/PRED-TMBB/input.jsp) was used to predict the extracellular loops, transmembrane domains and intercellular loops . A total of 54 peptides corresponding to the putative extracellular loop were synthesized and labeled with biotin (Additional file 2: Table S2). Ten serum samples were collected from PA-infected convalescent patients and another ten serum samples were collected from healthy donors. (See Additional file 3: Table S3).
A Luminex-based assay was set up to screen for dominant epitopes. Briefly, 30 μg of streptavidin (Thermo Fisher, Waltham, US) was covalently coupled to 2.5×106 beads according to the manufacturer’s instructions (Luminex, Austin, US). The beads were then incubated with biotin-tagged peptides (2 μg/ml) at 37 °C for 1 h. After washing with phosphate-buffered saline plus 0.1% Tween-20 (PBST), serum samples diluted 1:200 were added and incubated at 37 °C for 1 h. After the removal of the supernatant, the beads were washed with PBST. A phycoerythrin (PE)-labeled goat anti-human secondary antibody (Abcam, Cambridge, UK) at a 1:2500 dilution was added for incubation at 37 °C for 40 min. Finally, the fluorescence intensity of the beads was measured using a Luminex 200 instrument, and the results are expressed as the median fluorescence intensity (MFI). The MFI of beads without peptides was used as a control. Another hundred serum samples from patients recovered from PA infection were collected and used to verify the top 10 dominant epitopes. The methods were the same as those described above.
To evaluate the immunogenicity of the eight peptides (PA4554 D148-T172, PA4554 C167-W193, PA0958 A200-Q235, PA2398 T302-V331, PA2398 F636-G672, PA2398 K694-Q712, PA2398 Q737-K754, PA0165 R163-A174) in mice, these peptides were synthesized and conjugated to KLH (keyhole limpet hemocyanin) (Sigma, Milwaukee, US). The peptide–KLH conjugates formulated with the adjuvant Al(OH)3 were used for intramuscular immunization of BALB/c mice on day 0, day 14 and day 21. The injection volume for each animal was 500 μl, and each injection contained 100 μg of the conjugated preparation and 500 µg of Al(OH)3. PBS and Al(OH)3 were used as controls. Blood was collected via the tail vein seven days after the final immunization, and serum was isolated and stored at − 80 °C.
The reactivity of mouse sera against each peptide was determined by ELISA. The 96-well ELISA plates (Costar) were precoated with streptavidin (2 μg/ml) overnight at 4 °C. Blocking was performed with 1% bovine serum albumin (BSA) in PBST. Then, 2 μg/ml biotin-tagged peptide was added and incubated at 37 °C for 1 h. After the plates were washed with PBST, diluted serum samples (starting a dilution of 1:100 followed by 2-fold serial dilutions) were added for incubation at 37 °C for 1 h. Then, HRP-labeled goat anti-mouse IgG (Abcam) was added at a 1:5000 dilution for incubation at 37 °C for 45 min. The color was developed with TMB substrate solution (Beijing ZSGB-BIO) after washing, and the absorbance was measured at 450 nm. A sample was considered positive when the measured absorption value was more than 2.1-fold greater than the negative control (preimmune).
Preparation of PNPs, PNPs@M, and PNPs@M-Ep167-193
The PNPs (PLGA nanoparticles) were synthesized via a reported water-in-oil-in-water double emulsion method with slight modifications . In brief, PLGA (poly lactic-co-glycolic acid) (100 mg) was directly dissolved in methylene chloride (2 ml). Subsequently, the mixture was emulsified by sonication (35% amplitude, 2 min) using a Digital Sonifier S-250D (Branson Ultrasonic, Danbury, CT, US) in an ice bath. Next, the primary emulsion was immediately added to 10 ml of PVA (Polyvinyl alcohol) solution (3%, w/v) and sonicated for 3 min to form a double emulsion. The double emulsion was stirred overnight to remove the organic solvent. Then, the product was centrifuged at 12,000 rpm for 15 min and washed three times with deionized water.
Macrophage cell membrane encapsulate was prepared as described previously . The mouse macrophage cells (RAW264.7) were digested with trypsin, frozen at − 80 °C and thawed at room temperature. By repeated freeze–thaw three times, the membrane was collected by centrifugation at 14,000 rpm for 15 min, washed with PBS containing protease inhibitor and sonicated for 5 min. Subsequently, PNPs were mixed with the macrophage cell membrane (1:1 weight ratio of nanoparticles: membrane protein) . The mixture was sonicated in an ice bath for 3 min and maintained at 4 °C overnight. The PNPs@M (Macrophage membrane-coated PLGA nanoparticles) was finally collected by high-speed centrifugation at 12,000 rpm for 15 min. To obtain PNPs@M carrying Ep167-193 (PNPs@M-Ep167-193), DSPE-PEG (2000)-Ep167-193 was dissolved in disinfected water and mixed with the solution of PNPs@M. The mixture was reacted at room temperature for 1 h. The residual DSPE-PEG(2000)-Ep167-193 was eliminated by centrifugation at 12,000 rpm for 15 min.
The size and morphology of the nanoparticles were determined using a transmission electron microscope (Tecnai G2 F20 U-TWIN, FEI, Hillsboro, OR, US). The zeta-potential and size distribution were measured at room temperature using a Nano-ZS (Malvern, Worcestershire,UK). To confirm the membrane camouflage, PNPs@M was denatured and resolved via 12% SDS-PAGE. The gel was disassembled and proteins in the gel were stained for 1 to 2 h in Coomassie blue staining solution. Then the gel was destained with 10% acetic acid, which was changed every 30 min until the background is clear .
The toxicities of PNPs@M-Ep167-193 and PNPs@M on DC2.4 mouse dendritic cells and L929 mouse fibroblast cells were determined by the standard Cell Count Kit (CCK-8) assay. The cells were incubated with PNPs@M-Ep167-193 and PNPs@M at various concentrations (0, 25, 50, 100 and 200 μg/ml) for 24 h, 48 h and 72 h, respectively. Erythrocytes (300 μL) diluted in 0.9% NaCl solution were incubated with 1.2 ml of PNPs@M-Ep167-193 at 37 °C for 2 h. The absorbance of the supernatant was tested at 450 nm using a microplate reader. The experiments were conducted in triplicate and repeated twice. The biocompatibility of PNPs@M-Ep167-193 in vivo was assessed by a mouse experiment. The mice were cared for and treated as demonstrated in the preparation of PNPs@M-Ep167-193. On day 0, day 14 and day 21, the mice were immunized intramuscularly with 50 μg of PNPs@M-Ep167-193 (based on the concentration of Ep167-193). The mice were sacrificed 14 days after the third immunization, and their major organs were obtained by surgery. The pathological changes were observed with an Olympus DX51 optical microscope (Tokyo, JPN) after HE staining. The body temperature and body weight of the mice were monitored and recorded every day during the 35 days of observation.
Evaluation of the immunogenicity of PNPs@M-Ep167-193
A total of 20 BALB/c mice were randomly divided into four groups. On day 0, 14 and 21, the mice in each group were immunized with PNPs@M-Ep167-193 (50 μg), Ep167-193 (50 μg), PNPs@M (50 μg) or PBS. Seven days after the first, second and final immunization, blood was collected via the tail vein, and serum was isolated. The titers of total IgG and the subtypes against Ep167-193 in the sera were determined by ELISA as described above. HRP-labeled goat anti-mouse IgG, IgG1, IgG2a or IgG2b (Abcam) at a 1:5000 dilution was used as the secondary antibody.
Evaluation of protection conferred by immunization with PNPs@M-Ep167-193
A total of 15 mice were immunized with PBS, PNPs@M, Ep167-193 or PNPs@M-Ep167-193 as described above. Seven days after the last immunization, the mice in each group were intratracheally injected with a sublethal dose of PA XN-1 (1.3 × 106 CFU/mouse). Then, the infection was scored according to the breathing, piloerection, movement, nasal secretion and posture of the mice as described previously . The global score was recorded as unaffected (0–1), slightly affected (2–4), moderately affected (5–7), or severely affected (8–10). Mouse body weights were recorded every 24 h for 10 days.
The lungs of the mice were collected 24 h after the challenge and homogenized in 1 ml of sterilized PBS. Homogenates were serially diluted, plated on LB agar plates, and incubated overnight at 37 °C. Counts of viable PA XN-1 were determined by counting the colonies on the agar plates. Additionally, homogenates collected as described above were centrifuged, and the supernatants were used for cytokine analysis. The levels of TNF-α, IL-1β, IL-6 and IL-12 were measured using a Mouse ELISA Kit (Dakewei) according to the manufacturer’s instructions.
Twenty-four hours after the sublethal challenge, the lungs from mice in different groups were collected and fixed with 4% paraformaldehyde. Then, the samples were paraffin-embedded and cut into 4 μm section slices. The slices were stained with hematoxylin and eosin (HE) and viewed by light microscopy at 400×magnification. Each section was given disease scores in terms of the states of hemorrhage, edema, hyperemia, neutrophil infiltration, and destruction of bronchi structure by a pathologist in a blinded fashion according to a previously reported method . Each state was scored from 0 to 2 (0 = none, 2 = severe), and the final score of each section was the sum of the scores from the five states.
Data are shown as the mean ± standard error (SE). The significance of the differences was determined by unpaired parametric test (Student’s t test for two groups or one-way ANOVA for more than three groups). Bacterial burden was analyzed by the nonparametric Mann–Whitney test. IBM SPSS Statistics version 19.0 software (IBM Corp., Armonk, US) and Prism 8.0 software (GraphPad, US) were used to analyze the data. Significance was accepted when P < 0.05.