Animals
Female ICR mice aged 5–7 weeks were purchased from Hunan SJT Laboratory Animal Co., Ltd. (Changshang, China), and maintained under pathogen-free conditions in the laboratory animal room of the Parasitology Unit, Guangxi University, China. All mice were housed according to the Animal Ethics Procedures and Guidelines for the Use and Care of Laboratory Animals of the People’s Republic of China.
Preparation Of Esps
Adult F. gigantica parasites were obtained from the gallbladders of freshly killed swamp buffaloes at local abattoirs. Adult parasites were washed three times with phosphate-buffered saline (PBS), then cultured in PBS (1 worm/1 mL of PBS) at 37°C for 60 min to remove host contaminants (e.g., bile, liver tissue, and blood) from the guts. Parasites were then incubated in RPMI 1640 culture medium (GIBCO, Grand Island, NY, USA) containing 0.1% glucose, 100 U penicillin, and 100 µg/ml streptomycin (Solarbio, Beijing, China), at 1 worm/mL for 5 h at 37°C. The supernatant was collected and used for the next step.
Ev Isolation And Purification
EVs were isolated from F. gigantica culture media according to the differential centrifugation protocol reported by Marcilla [22] with some modifications. Briefly, the parasite culture media was centrifuged at 300 × g for 15 min and then at 7000 × g for 30 min to remove eggs, cells, and large debris. The resulting supernatant was centrifuged at 2,000 × g for 45 min at 4°C. The resulting supernatant was centrifuged at 15,000 × g for 60 min at 4°C to obtain 15k EVs (mainly microvesicles). ESP supernatants were then filtered, using a 0.22 µm ultrafiltration membrane, and centrifuged at 100,000 × g for 90 min at 4°C to pellet the 100k EVs (exosomes like EVs). Both the 15k and 100k EV pellets were washed twice with a large volume of PBS at the same high speed. The resulting supernatant was concentrated using Amicon® Ultra-15 3 kDa NMWCO tubes (Millipore, Darmstadt, Germany) at 5,000 × g at 4°C, and it was then washed three times with PBS. Protein contents were quantified by the bicinchoninic acid assay (BCA) kit (CWBIO, Beijing, China) and then stored at − 80°C for further studies.
Transmission Electron Microscopy (Tem) Analysis Of Ev Samples
The 15K EVs and 100K EVs pellets were added on formvar-coated copper grids (Zhongjingkeyi Technology, Beijing, China) and stained with 2% phosphotungstic acid (Solarbio, Beijing, China) for 1 min and at room temperature. EVs on the grids were imaged using a HITACHI-H7700TEM (HITACHI, Tokyo, Japan) and exposed at 100 kV.
Nanoparticle Tracking Analysis (Nta)
To measure the size distribution of particles in the EV samples, NTA was carried out using a ZETASIZER NANO ZSP (Malvern, UK) to capture and analyze the data. EVs were diluted by PBS and then loaded into the sample chamber. For each sample, three measurements were performed and the data were analyzed.
Proteomic analysis of F. gigantica 100K EVs
Proteomic analysis was done on F. gigantica 100K EVs and two independently separated samples were assayed. The 100K EVs protein samples were digested with trypsin as described by Wu [23], with some modifications. According to the quantitative results, 3 µg of the digested peptides was analyzed by LC-MS/MS (Thermo Scientific, Waltham, MA, USA). Digested peptides pre-equilibrated with buffer A (0.1% v/v formic acid aqueous solution) were added to a 5 µm-C18 EASY column (2 cm × 100 µm, Thermo Scientific) and separated with a linear gradient of buffer B (0.1% v/v formic acid in 84% v/v acetonitrile aqueous solution) on a 3 µm-C18 EASY column (75 µm*100 mm 3 µm-C18, Thermo Scientific). The Easy nLC system (Thermo Scientific) was used to deliver buffer A and buffer B with a linear gradient of 0–55% (0–110 min), 55–100% (110–115 min), and maintained at 100% (115–120 min), at a flow rate of 300 nL/min. MS scan data were acquired using Q-Exactive (Thermo Scientific), and the 20 most abundant precursor ions from the survey scan (300–1800 m/z) were selected for higher-energy collisional dissociation fragmentation. The automatic gain control (AGC) target was set to 3e6, maximum inject time (IT) was 50 ms, and dynamic exclusion duration was 60.0 s. Using predictive automatic gain control, scans were acquired at a resolution of 70,000 at m/z 200 with a dynamic exclusion duration of 60 s, and the target value was determined for full scans. The resolution for the high-energy collisional dissociation spectra was set to 17,500 at m/z 200, with an isolation window at 2 m/z. The normalized collision energy was set at 27 eV, and the underfill ratio was defined as 0.1%.
The FASTA database was from F. hepatica (consisting of 26,283 entries), and F. gigantica (consisting of 13,094 entries) obtained from UniProt (https://www.uniprot.org/) were used for the analysis of the EV mass spectrometry data through Proteome Discoverer 2.2 (Thermo Scientific). Only proteins with at least three peptides were considered to be positively identified.
Protein Expression And Purification Of Rfg-hsp70
The full-length sequence of heat shock protein (HSP70) (GenBank: ABS52703.1) of F. gigantica was obtained from the GenBank database. To produce HSP70, the F. gigantica HSP70 gene sequence was synthesized, after codon optimization, then cloned into a pET28a(+) vector using BamHI and XhoI cloning sites. The expression pET28a(+) vector was transformed into Escherichia coli BL21 (DE3) competent cells and induced with 0.1 mM isopropylthio-β-galactoside (IPTG) (Solarbio, Beijing, China) at 30°C for 8 h. Recombinant Fg-HSP70 were purified by affinity chromatography using a His-tag Protein Purification Kit (Beyotime, China). New Zealand White rabbits were immunized with rFg-hsp70 to produce polyclonal antibodies against rFg-hsp70, using the method described by Anuracpreeda et al. [24].
Sds-page And Western Blot (Wb) Analysis
SDS-PAGE and Western blot (WB) analysis
The 15K and 100K EVs (20 ug amounts) were loaded and separated using 15% SDS-PAGE gel, and proteins were transferred to 0.45 µm polyvinylidene difluoride (PVDF) membranes (Merck Millipore, USA). The membranes were blocked with blocking solution (PBST: PBS, 0.05% Tween-20, 5% non-fat powdered milk) for 4 h at room temperature, followed by overnight incubation at 4°C with Rabbit anti-human CD63 monoclonal antibody (dilution 1:500; Abcam, USA) and Rabbit polyclonal antibodies raised against F. gigantic leucine amino peptidase (LAP), Heat shock protein 70 (HSP70), and 14-3-3 epsilon (dilution 1:200). Then the membranes were washed five times for 10 min each with PBST. The membranes were further incubated for 4 h at 4°C with the secondary Goat Anti-Mouse IgG (dilution 1:5,000; Abcam, USA) antibody conjugated with horseradish peroxidase (HRP). After five washes of 10 min each with PBST for chemiluminescence imaging with a BeyoECL Plus kit (Beyotime, Nantong, China), the membranes were visualized using ImageQuant LAS 500 (Cytiva, USA).
Vaccination Studies On Experimental Animals And Vaccination Protocol
Forty 7-week-old female ICR mice were randomly divided into five groups (8 mice/group): (1) non-immunized and uninfected group (Blank group), (2) immunized with PBS plus Alum Adjuvant (Thermo, Rockford, IL, USA) and infected group (Alum + PBS group), (3) immunized with 200 µg of ESP plus Alum Adjuvant and infected group (Alum + ESP group), (4) immunized with 200 µg of 15K EVs plus Alum Adjuvant and infected group (Alum + 15K EVs group), (5) immunized with 200 µg of 100K EVs plus Alum Adjuvant and infected group (Alum + 100K EVs group), and (6) immunized with 200 µg of rFgHSP70 plus Alum Adjuvant and infected group (Alum + rHSP70 group). Mice were maintained in the laboratory animal room of Parasitology Unit, Guangxi University, China. All animal experimental procedures were performed according to the National Institutes of Health guide for the care and use of laboratory animals (NIH Publication No. 8023, revised 1978). Mice were kept in steel cages in an air-conditioned room at 23 ± 2°C, with a 12:12 h (L:D) photoperiod and 50–60% relative humidity. Vaccines were injected intraperitoneally on days 0, 14, and 28. Treated mice were challenged with 15 F. gigantica metacercariae, via oral administration, on day 42. At 28 d post-challenge, mice were sacrificed using CO2, and blood samples were collected for serum by heart puncture. Their peritoneal cavities were opened and washed with PBS. Whole livers were collected to determine the number of F. gigantica flukes (Fig. 4).
Worm Recoveries And Percent Protection
The percent reduction of F. gigantica worm recovery was calculated as previously described [24]. Worm reduction (%) = ((A–B) / A) × 100% (A = worm burden immunized with PBS plus Alum Adjuvant group, and B = worm burden in the challenged immunized group).
Antiparasite Igg1 And Igg2a/c Elisas
Specific IgG, IgG1, and IgG2a against Fg-15k EVs, Fg-100k EVs, or Fg-ESP were carried out by indirect ELISA as described previously [3]. The serum IgG, IgG1, and IgG2a antibody response against F. gigantica 15K EVs, 100K EVs, and ES (depleted of EVs) was measured, in triplicate, for each group by indirect ELISA.
Statistical Analyses
Results are represented as means ± standard error of the mean (SEM), and analyses were performed using Graphpad Prism Software Version 6.01 (GraphPad Software Inc., San Diego, CA, USA). Statistical analysis was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test or Student’s t test. Linear regression analysis between the IgG, IgG1, IgG2a, and the numbers of flukes recovered at termination in the ESP-immunized, 15K EV-immunized, 100K EVs-immunized, rFgHSP70-immunized, and adjuvant-infected control were analyzed by Pearson’s r correlation test using Origin software (Guangxi University, China). P values of < 0.05 (*), < 0.01 (**), and < 0.001 (***) indicated statistical significance.