Materials
PLGA (lactide: glycolide = 50:50; MW = 30,000–60,000), polyvinyl alcohol (PVA; MW = 85 000-124 000, 99% hydrolyzed), HRP-labeled goat anti-mouse IgG, Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma-Aldrich (St Louis, MO). The human lung adenocarcinoma epithelial cell line A549 was obtained from American Type Culture Collection (ATCC). RPMI-1640 medium, fetal bovine serum (FBS) and antibiotic-antimycotic were all purchased from Invitrogen (Carlsbad, CA). The Celltiter96 CCK8 Cell Proliferation Assay kit was purchased from KeyGEN BioTECH (Nanjing, China). Fc blocking antibody, Mouse I-Ab APC, PerCP-Cy5.5 anti-mouse CD11c, PE anti-mouse F4/80, FITC anti-mouse CD11b, PE anti-mouse CD3 and APC anti-mouse CD19 were all bought from BD-Biosciences (San Diego, CA). DNA ligase, DNA polymerase (Taq enzyme), restriction enzymes BamHI and XhoI were purchased from American Thermo Company.
A. baumannii ATCC19606 strain was obtained from American Type Culture Collection (ATCC). Three clinical A. baumannii strains were collected from the Second Affiliated Hospital of Nanjing Medical University. All clinical A. baumannii strains were confirmed to be multi-drug resistant (MDR) strains by drug sensitivity experiments (Additional file 1: Table S1) according to clinical and laboratory standards institute (CLSI) M100. The E. coli BL21 (DE3) and the plasmid pET28a (+) used in the study were purchased from Novagen company (Beijing, China) and kept in our laboratory. For all experiments, unless otherwise stated, bacteria were grown on Luria-Bertani (LB: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride) agar plates or in LB broth at 37℃.
Animals
All animal experiments were performed using 6 to 8 weeks old female BALB/c mice purchased from Shanghai Super - B&K laboratory animal Corp. Ltd. (Shanghai, China). The mice were raised under specific pathogen-free (SPF) conditions at ambient temperature of 25 ℃ and provided with sterile food and water ad libitum. The animal experimental procedures were approved by the Ethics Committee of Animal Care and Welfare, Nanjing Medical University (Nanjing, China) (Approval Number: IACUC-1904044). All efforts were made to minimize animal suffering.
Expression and purification of Omp22
The gene encoding Omp22 was obtained through PCR amplification, using the genomic DNA of A. baumannii ATCC 17978 strain as the template. The primers were Omp22-F (5′ CAA GGA TCC CTG GGC GGC GTT GAA TAT G 3′) and Omp22-R (5′ CAC AAG CTT TTA CTG TTT CGC GTA AAT G 3′). The PCR products was digested using the BamHI and XhoI enzymes and ligated into the plasmid pET28a. The recombinant plasmid pET28a-omp22 was transfected into E. coli BL21 (DE3). The recombinant protein Omp22 was obtained through induced expression and purified. After eluted with elution buffer (1 M imidazole in binding buffer), the collected fractions were analyzed with SDS-PAGE.
Prediction and Identification of T-cell epitopes and B-cell epitopes
The physicochemical properties of A. baumannii Omp22 protein was analyzed and all possible dominant B-cell and T-cell epitopes were predicted using the immuno-informatics approach. The B-cell epitopes were predicted using bioinformatics software OptimumAntigen™ Design Tool (GenScript, China). Four candidate B-cell epitopes were predicted according to their secondary structure, surface accessibility, hydrophilicity, flexibility, and antigenic index. When predicting T-cell epitopes, The IEDB (Immune Epitope Database Analysis Resource) (https://tools.iedb.org/mhci/) was used and four T-cell epitopes were stored based on their scores[34, 35]. The predicted B-cell and T-cell epitope peptides were chemically synthesized by Jiangsu GenScript Biotechnology Co. Ltd.
Twelve female BALB/c mice (6 to 8 week) were randomly divided into two groups, Omp22 immunized group and phosphate-buffered saline (PBS) control group. Mice were subcutaneously injected with 100 µg recombinant Omp22 (1 µg/µl in PBS) or an equal volume of PBS for three times with two-week interval. In the Omp22 vaccine group, Freund’s adjuvant was added to enhance the immune effect. One week after the last immunization, serum of each mouse was collected to detect B-cell epitope specific antigen by indirect ELISA. Splenocytes were isolated from vaccinated mice and adjusted to a concentration of 1 × 106 cells/ml, and 200 µl of the cell suspension was added to each well of a 96-well plate and stimulated with 20 µg/ml of candidate T-cell epitopes. After incubated for 72 h, supernatants were collected and levels of gamma interferon (IFN-γ) were measured using mouse IFN-γ ELISA kits. Based on the above analysis, three B-cell epitopes (amino sequences: NIPLSQARAQSVKNY; YATLDKVAQTL and SVQLIMP) and two T-cell epitopes (amino sequences: VPSSRIDAQGY; TFDTNKSNIKP) from Omp22 were selected to design the multi-epitope protein.
Design and synthesis of rOmp22
Three optimal B-cell epitopes and two T-cell epitopes were connected in series by 6-aminocaproic acid. A multi-epitope peptide rOmp22 of 59aa, with a molecular weight of 6536.4 Da was chemically synthesized and identified by liquid chromatography and mass spectrometry. The synthesis and identification of rOmp22 was completed by Jiangsu GenScript Biotechnology Co. Ltd.
Preparation of nanoparticles
CS-PLGA NPs were prepared using a modified water/oil/water double emulsion evaporation technique[36]. Briefly, 2 mg of rOmp22 dissolved in PBS containing 0.003% sodium alginate was the inner water phase, 80 mg of PLGA (lactide: glycolide = 50:50; MW = 30000–60000) (Sigma-Aldrich) was dissolved in 2 ml dichloromethane (DCM) as the organic phase. 2% (w/V) polyvinyl alcohol (PVA; MW = 85000–124000, 99% hydrolyzed; Sigma-Aldrich) solution including 0.2% chitosan (MW = 50000; Sigma-Aldrich) was prepared as the external aqueous phase. The emulsion was stirred at room temperature overnight. The nanoparticles were obtained by ultracentrifugation, then washed three times with deionized water to remove excess polyvinyl alcohol and rOmp22, lyophilized to obtain CS-PLGA-rOmp22. An equivalent volume of PBS as used for rOmp22 was similarly encapsulated in CS-PLGA to obtain CS-PLGA-PBS to serve as a negative control. All lyophilized nanoparticles were stored at -80℃ until used.
Encapsulation efficiency and peptide loading level
The encapsulation efficiency of rOmp22 in CS-PLGA was measured using HPLC (Agilent, USA) by quantitating rOmp22 in supernatant after ultracentrifugation. The rOmp22 encapsulation efficiency (EE) and the peptide loading capacity (LC) were calculated using the following formulas:
EE = (A-B)/ A × 100
LC = (A-B)/ C × 100
Where A is the total amount of rOmp22, B is the free amount of rOmp22, and C is the CS-PLGA-rOmp22 weight. These measurements were performed three times.
Particle size and zeta potential
The particle size, polydispersity index (PDI) and zeta potential were measured by particle size analyzer (Anton Paar, Graz, Austria). CS-PLGA-rOmp22 or CS-PLGA-PBS was suspended in filtered distilled water, sonicated, and placed in a cuvette to measure size and zeta potential. Each sample was measured three times and reported as the mean of triplicates for size (diameter in nanometers) and zeta potential (millivolt). These experiments were conducted at least three times.
Transmission electron microscopy (TEM)
The morphology of CS-PLGA-rOmp22 and CS-PLGA-PBS were observed using high-resolution TEM (Hitachi, HT7700 Exalens). One drop of the complex was deposited on the copper grid (carbon-coated copper grid, 200 mesh). After adding phosphotungstic acid, the grids were dried for 10 min prior to TEM analysis.
In vitro peptide release
The release of the rOmp22 peptide from CS-PLGA was determined following the method of Bouissou et al[37]. Briefly, CS-PLGA-rOmp22 NPs were suspended in normal saline (NS). The suspensions were incubated at 37℃ and at various time intervals (30 min, 1, 2, 4, 6, 12, 24, 48 and 72 h), supernatants were collected by centrifugation. The released peptide in the supernatants was measured using HPLC.
Cytotoxicity studies
The cytotoxicity of rOmp22, CS-PLGA-PBS and CS-PLGA-rOmp22 to human lung adenocarcinoma epithelial cell line A549 (ATCC) was detected by Cell Counting Kit-8 (CCK8) assay. In cell viability assay, A549 cell were seeded at 1 × 105 cells/well in 96-well plates in 100 µl of medium and incubated overnight at 37℃ in a humidified atmosphere with 5% CO2 to allow cell attachment. Then the cells were incubated with different concentrations of rOmp22 (1.25-80 µg/ml), CS-PLGA-PBS (12.5–800 µg/ml) or CS-PLGA-rOmp22 (12.5–800 µg/ml) for 6, 24 and 48 h. And then, 10 µl of CCK8 solution was added to the culture medium and incubated for an additional 2 h. Optical density (OD) values were measured at 450 nm wavelength using a microplate reader (Bio-Rad iMark).
Mouse immunization
BALB/c mice were randomly divided into four groups: CS-PLGA-rOmp22 treated group, CS-PLGA-PBS treated group, rOmp22-immunized group and an adjuvant-treated group. Mice in the CS-PLGA-rOmp22 group were immunized subcutaneously with 40 µg/200 µl of encapsulated rOmp22 in NS, and those in the CS-PLGA-PBS group were immunized with an equivalent weight of CS-PLGA-PBS NPs. Each mouse in the rOmp22 group was immunized with 40 µg non-encapsulated rOmp22 in 100 µl NS formulated with an equal volume of Freund’s complete adjuvant (Sigma), and boosted with the same dose of rOmp22 in 100 µl NS formulated with Freund’s incomplete adjuvant on day 14 and 28. In the adjuvant-treated group, each mouse was injected subcutaneously with 100 µl NS formulated with equal volume of Freund’s complete adjuvant on day 0 or Freund’s incomplete adjuvant on days 14 and 28. Six mice were randomly selected from each group for immunological assay. The other animals were used for challenge test two weeks after the third immunization.
Antibody titer measurement with ELISA
Multi-epitope peptide rOmp22 was first diluted to an optimal concentration (10 µg/ml) to coat a 96-well plate. The resulting solution was then added into each well (100 µl per well) and incubated for 12–18 h at 4℃. After washing five times with PBS plus 0.05% Tween 20 (PBST), 200 µl of 2% bovine serum albumin (BSA) was added to each well and incubated for 2 h at 37℃ to block the unoccupied sites. After washing, serial dilutions of the pooled serum in each group ranging from 1:200 to 1:51200 were added to the wells in triplicate and incubated at 37℃ for 2 h. Plates were washed 5 times as described above. And then 100 µl per well of Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (diluted 1:10000) was added and the plates were incubated for 1 h. Plates were then washed 5 times and incubated with 100 µl per well of 3,3’,5,5’-tetramethylbenzidine solution (TMB) as substrate until a desired absorbance was reached. The reaction was stopped by the addition of 100 µl of 2 M H2SO4. The optical density of the samples was measured at 450 nm using an ELISA plate reader. The endpoint titer was defined as the highest dilution of which the OD at 450 nm was at least 0.1 above that of the background well.
Splenocyte stimulation and cytokine secretion
Splenocytes were isolated as described previously from vaccinated mice seven days after the last immunization[10]. Splenocytes were adjusted to a concentration of 1 × 106 cells/ml, and 200 µl of the cell suspension was added to each well of a 96-well plate and either stimulated with rOmp22 (20 µg/ml) or left unstimulated. After incubated for 72 h, supernatants were collected and levels of gamma interferon (IFN-γ) and Interleukin 4 (IL-4) were detected using mouse IFN-γ and IL-4 ELISA kits.
Flow cytometry analysis
Single spleen cell suspensions and draining lymph nodes cell suspensions were obtained from mice one week after the last immunization. Cells (1 × 106/ml) were blocked with Fc blocking antibody (BD Bioscience) in fluorescent-activated cell sorting (FACS) buffer (phosphate buffered saline, 1.0% fetal bovine serum) for 15 min at 4℃. The cells were washed and stained with fluorochrome-conjugated antibodies against lymphocyte surface receptors, APC-Mouse I-Ab, PerCP-Cy5.5-CD11c, PE-F4/80, FITC-CD11b, PE-CD3 and APC-CD19 (BD Biosciences) for 30 min at 4 °C. The cells were then washed and fixed with 2% paraformaldehyde solution for 20 min at 4 °C. Data were acquired on a BD FACS Canto II flow cytometer (BD Bioscience) with at least 1 × 105 events for each sample and analyzed using FlowJo software (Tree Star Inc, Ashland, OR, USA).
Establishment of pneumonia models
A. baumannii ATCC19606 strain and 3 clinical A. baumannii strains (CS-MDR-AB, CRAB and PDR-AB) were grown in LB broth to the late-logarithmic phase at 37℃/150 rpm. Cells were harvested by centrifugation at 4000 g for 10 min, washed and resuspended in PBS and mixed with porcine mucin (Sigma-Aldrich) to a final concentration of 5% mucin. Desired CFU/ml was obtained by appropriate dilutions and the final concentration was quantified by plating serial dilutions onto LB agar plates. The mice were anaesthetized with intraperitoneal (i. p.) injection of pentobarbital sodium, placed in a supine position and their trachea were exposed surgically. Lethal doses of ATCC19606 (2 × 108 CFU) and three clinical A. baumannii strains, CS-MDR-AB (1 × 109 CFU), CRAB (5 × 108 CFU) or PDR-AB (5 × 108 CFU) in a total volume of 100 µl was intra-tracheally to mice to induce acute pneumonia. The incised area was sealed with sterile surgical sutures. The mice were monitored for 7 days, body weight, clinical score and survival number from each group were recorded every day.
Bacterial load assessment in blood and lung
Blood samples were collected from six mice in each group at 24 h post-challenge. To determine the bacterial loads in blood, samples were serially diluted and plated on blood agar plates. After taking blood, the mice were killed, lungs were removed aseptically, weighed, and homogenized. Serial dilutions of tissue homogenates were plated onto blood agar plate. Bacterial CFUs were enumerated after 24 hours of incubation at 37℃.
Histopathological examination
Lungs were removed under aseptic conditions and fixed in 4% formalin. Histopathological examination of the section after embedding in paraffin was observed under microscope after staining with haematoxylin-eosin (HE). Lung injury was estimated by the percentage of the lesion area in the total lung area using an ImagePro macro.
Statistical analyses
Statistical analyses were performed using the Statistical Package of Social Sciences (SPSS, version 23.0; SPSS Inc., Chicago, IL) and Graphpad Prism (version 6.0; Graphpad software Inc., La Jolla, CA). All data were expressed as Mean ± SD. The one-way analysis of variance (ANOVA) was used for multiple comparisons, followed by Bonferroni’s post hoc test. Survival data were compared using the log-rank test. A P value of < 0.05 was considered significant.