Animals and parasites
Female C57BL/6 mice (6–8 weeks old) were purchased from the Changsheng Experimental Animal Center (Changchun, China) and maintained under specific pathogen-free conditions at the National Experimental Teaching Demonstration Center of Jilin University (Changchun, China). The food and water provided were sterile. All animal experimental procedures were performed in strict accordance with the approval of the Animal Welfare and Research Ethics Committee at Jilin University. N. caninum tachyzoites (Nc-1 strain) were maintained by serial passage in Vero cells in RPMI-1640 medium, and free tachyzoites were obtained and harvested from Vero cells as described in a previous study[7, 8].
N. caninum EV preparation
N. caninum EVs were purified as previously described. Briefly, free tachyzoites were collected using Percoll and cultured in exosome-depleted medium for 24 h. Parasite culture supernatants were collected and centrifuged to remove the parasites and debris. Finally, the supernatant was passed through a 0.22-μm syringe filter (Millipore, Billerica, USA), followed by further ultracentrifugation (Hitachi Micro Ultracentrifuge, Japan) at 100,000×g for 70 min at 4°C to spin down the expected N. caninum EVs (NEVs). The NEV-rich fraction was washed twice and then resuspended in PBS. Protein concentrations were measured using a BCA Protein Assay Kit (Thermo Scientific, Waltham, USA) and then stored at -80°C or directly used in additional experiments.
Expression and purification of recombinant Nc14-3-3 protein
The Nc14-3-3 PCR product was cloned into a pGEX-4T-1 vector, which has a GST tag, and the recombinant plasmid pGEX-Nc14-3-3 was transformed into the E. coli expression strain Rosetta DE3a (TIANGEN, Beijing, China). Flutathione S-transferase (GST) fusion protein expression was induced with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and purified with Proteinlso® GST Resin (TransGen Biotech, Beijing, China), as described previously. The induced expression of pGEX-4T-1 empty vector and purified GST-tagged protein were used as controls.
Mice immunization and challenge
To assess the immunogenicity of Nc14-3-3, female C57BL/6 mice were randomly divided into five groups (16/group). NEVs were dissolved in sterile phosphate-buffered saline (PBS) to a final concentration of 1 μg/μl, and 50 μg NEVs or PBS alone (each 50 μl) were injected into mice through the tail vein. Mice were subcutaneously immunized with recombinant Nc14-3-3 protein or GST protein (50 μg) emulsified with Freund’s adjuvant (Sigma, St. Louis, USA). The antigens were intramuscularly administered three times at 14-day intervals, blood samples of mice were collected from the tail vein plexus on the day before each vaccination, and the sera were obtained and stored at −20°C for ELISA. Two weeks after the last injection, each mouse was challenged with a dose of 2×107 Nc-1 tachyzoites, and the survival time, body weight and clinical observations in the mice were observed and recorded every day by the same person at similar time points.
Determination of serum antibody and cytokine levels
The levels of antibodies in mouse sera were detected by indirect enzyme-linked immunosorbent assays (ELISA), as previously described . Briefly, each well of 96-well plates (Corning Costar, Cambridge, MA, USA) were coated with 2 μg N. caninum lysate antigen (NLA) in 100 μl of carbonate buffer (150 mM Na2CO3, 349 mM NaHCO3, pH 9.6) and incubated at 4°C overnight. After three washes with PBST, the plates were blocked with 3% bovine serum albumin (BSA) for 2 h at 37°C and subsequently incubated with mouse sera diluted in PBST (1:100) for 2 h at 37°C. After three washes, the wells were incubated with 100 μl HRP-labelled secondary antibody (IgG, IgG1 or IgG2a, 1:2,000 dilution) (Proteintech, Wuhan, China) for 1 h at 37°C. The reaction was detected by TMB (Beyotime, Shanghai, China) for 15 min at 37°C and stopped by 2 M H2SO4. The absorbance was measured at 450 nm with an ELISA reader.
To evaluate the concentration of cytokines in serum samples, two weeks after the final immunization, six mice were sacrificed, blood was collected from the eyeball, and sera were obtained for cytokine measurements. Cytokine ELISA Ready-SET-Go kits (eBioscience, San Diego, CA, USA) were used to detect IL-12p40, IFN-γ, IL-10 and IL-4 levels according to the manufacturer’s instructions. The assays were read at 450 nm, and the optical density (OD) values were converted to pg/ml by extrapolation using a standard curve.
Flow cytometry analysis of T cell subpopulations
Flow cytometry was used to analyse the percentages of T cell subpopulations in spleens of mice in the experimental groups. The spleens were obtained two weeks after the final immunization from mice (n=6) in each group, and a flow cytometry assay was performed as previously described. Briefly, 1×106 splenocytes were suspended in 100 μl pre-cooled PBS and incubated with 0.5 μg anti-mouse CD3-PerCP, 0.25 μg anti-mouse CD4-PE and 0.25 μg anti-mouse CD8-APC (all from BioLegend) at 4 °C for 30 min in the dark. Then, the cells were washed twice with pre-cooled PBS, resuspended in PBS and analysed with a FACSAria flow cytometer (BD Biosciences) with 20,000 total events/sample. Data were analysed by FlowJo software (Tree Star Inc.).
Quantification of the parasite burden by qPCR
Two weeks after the last immunization, each mouse was challenged with 2×107 Nc-1 tachyzoites. At 5 days post-infection, infected mice were euthanized, and the heart, liver, spleen, hung, kidney, and brain were harvested and stored at -40°C. Parasite replication in the various tissues was monitored by real-time quantitative PCR (qPCR) analysis of parasite DNA, as previously described. Briefly, the tissues were homogenized and used for DNA extraction (TIANGEN, Beijing, China). Five hundred nanograms of extracted DNA from one sample was amplified with the Nc5 sequence of N. caninum (forward: 5’-ACTGGAGGCACGCTGAACAC-3’, reverse: 5’-AACAATGCTTCGCAAGAGGAA-3’) using FastStart Universal SYBR Green Master Mix. To quantify the number of parasites, a standard curve was generated by amplifying 10-fold dilutions of 2.3×108N. caninum tachyzoites in separate reactions.
Pathological changes were observed by H&E staining, and fresh tissue was fixed with 10% neutral buffered formalin and routinely processed in paraffin. Fixed paraffin-embedded tissues were sectioned at 3-4 µm and stained with haematoxylin and eosin (H&E).
Data are expressed as the mean ± standard deviation (SD), and means were compared by one-way analysis of variance using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). All graphs were generated with GraphPad Prism 7.00, and independent experiments were performed with at least three technical replicates.