Molecular characterization of S. japonicum exosome-like vesicles reveals their regulatory roles in parasite-host interactions

Secreted extracellular vesicles play an important role in pathogen-host interactions. Increased knowledge of schistosome extracellular vesicles could provide insights into schistosome-host interactions and enable the development of novel intervention strategies to inhibit parasitic processes and lessen disease transmission. Here, we describe biochemical characterization of Schistosoma japonicum exosome-like vesicles (S. japonicum EVs). A total of 403 proteins were identified in S. japonicum EVs, and bioinformatics analyses indicated that these proteins were mainly involved in binding, catalytic activity, and translation regulatory activity. Next, we characterized the population of small RNAs associated with S. japonicum EVs. Further studies demonstrated that mammalian cells could internalize S. japonicum EVs and transfer their cargo miRNAs to recipient cells. Additionally, we found that a specific miRNA, likely originating from a final host, ocu-miR-191–5p, is also associated with S. japonicum EVs. Overall, our findings demonstrate that S. japonicum EVs could be implicated in the pathogenesis of schistosomiasis via a mechanism involving the transfer of their cargo miRNAs to hosts. Our findings provide novel insights into the mechanisms of schistosome-host interactions.

elongation factor 1-gamma, twinfilin-1, tubulin gamma-1 chain, actin-related protein 2/3 complex subunit 4, et al. (Supplementary Table S1). In addition, 59 proteins (14.64%) were assigned to Kyoto Encyclopedia of Genes and Genomes analysis, and these assigned proteins are mainly involved in the integrin signaling pathway, CCKR signaling map, ubiquitin proteasome pathway, cytoskeletal regulation by Rho GTPase, disease-related pathways (Huntington disease and Parkinson disease), and inflammation mediated by chemokine and cytokine signaling pathways (Supplementary Table S2).

Identification of small RNAs associated with S. japonicum EVs. Small RNAs incorporated into
exosomes play a regulatory role in exosome-mediated cell-cell communication. Therefore, we identified the small RNA population associated with S. japonicum EVs using Solexa deep sequencing. The length distribution of S. japonicum EV-associated small RNAs ranged from 13 nt to 27 nt (Fig. 4a,b) and rRNAs, repeat associated small RNAs, and miRNAs were the dominant class of small RNAs (Fig. 4c). We found that 15 known S. japonicum miRNAs (cut-off reads > 100) were present in S. japonicum EVs libraries (Table 2 and Supplementary Dataset 3), including two miRNAs (Bantam and miR-10) identified in the plasma of S. japonicum infected hosts in our previous study 24 . We also predicted 19 novel miRNAs using Mireap (Supplementary Dataset 4). In addition, there are 4 miRNAs associated with S. japonicum EVs (cut-off reads > 100) homologous to miRNAs in ExoCarta database (Supplementary Table S4). Interestingly, we found that there were 1,284,966 reads (3.4%, 2,540 unique small RNAs) specifically mapped to the rabbit genome, including one miRNA, ocu-miR-191-5p, with a relatively high number of reads (4,548 reads) ( Fig. 4d and Table 2). Next, we used stem-loop based qRT-PCR to further verify the present of ocu-miR-191-5p in the RNAs isolated from S. japonicum EVs (Fig. 4e). These findings suggested that S. japonicum EVs could carry both host and schistosome factors that could be involved in the pathogenesis of schistosomiasis and/or the regulation of schistosome development.

Analyses of antibody responses to S. japonicum EVs. To evaluate immune responses against S. japonicum
EVs in a final host, we used enzyme-linked immunosorbent assay (ELISA) and Western blot to examine immunological recognition of S. japonicum EVs. As shown in Fig. 5, sera from infected rabbits indicated significant detection of S. japonicum EV proteins as compared with that of controls (Fig. 5a). Western blot analyses showed that sera from infected rabbits recognized multiple EV bands, whereas only a few bands were observed when applying uninfected serum (Fig. 5b). Reactive bands in the EVs were also different from those present in the 293 T cell preparations, which were detected with both infected serum pools and uninfected pools suggesting that infected sera could specifically recognize some proteins from S. japonicum EVs. These results indicate that schistosomes secrete EVs into the circulation of the final host and that secreted S. japonicum EVs and their protein components could be potential biomarkers for schistosomiasis diagnosis.

Internalization of S. japonicum EVs in mammalian cells.
Exosome cargo, especially miRNAs has been shown to play a regulatory role in host-pathogen interactions. To reveal potential roles of S. japonicum EVs in host-pathogen interactions, we labeled S. japonicum EVs with the lipid dye PKH67 and incubated the labeled exosomes with mouse liver cells (NCTC clone 1469 cells) in vitro. S. japonicum EVs were internalized by the NCTC clone 1469 cells (Fig. 6a). Further analyses demonstrated that S. japonicum EV associated miRNAs (Bantam and miR-10) were significantly detected in the RNA isolated form cells treated with labeled S. japonicum EVs (Fig. 6b), indicating that the miRNA cargo of S. japonicum EVs can transfer to recipient cells.
qRT-PCR analysis of the expression of potential target mRNAs of S. japonicum EVs associated miRNA in mice. To determine potential regulatory roles of the cargo miRNA of S. japonicum EVs in host cells, we performed bioinformatics analyses to predict the target mRNAs for a specific S. japonicum associated miRNA, Bantam miRNA. Three murine genes (Gins4, Tysnd1, and Utp3) were shown to be potential targets of Bantam miRNA in mice (Fig. 7a). We analyzed the expressions of these three mRNAs in murine liver cells (NCTC clone 1469 cells) treated with S. japonicum EVs. All three genes were significantly (P < 0.05) down-regulated (Fig. 7b). To further corroborate these results, we determined the expressions of these three genes in the livers of mice infected with S. japonicum cercariae at 25 and 35 d post infection (dpi). We observed significantly (P < 0.05) down-regulated expression of these genes in animal level (Fig. 7c). Overall, these results indicated that uptake of S. japonicum secreted EVs could potentially regulate mouse genes via S. japonicum originating miRNAs.

Discussion
Exosomes have been shown to act as signals for the membranes of target cells, participate in pathogen dissemination, and exert effects in host cells, including those of the host immune system 15,25,26 . However, knowledge of S. japonicum EVs is still limited, although a recent study indicated the existence of EVs derived from S. japonicum adult worms 21 . The proteins and miRNA cargo in S. japonicum EVs remain uncharacterized. It also remains unknown whether S. japonicum EVs can be internalized by mammalian cells and transfer miRNAs to recipient cells. In the present study, we developed a protocol to isolate S. japonicum EVs and then characterized the protein components of S. japonicum EVs and their associated small RNA population. We demonstrated that S. japonicum EVs can be internalized by mammalian cells and transfer their associated miRNAs to recipient cells. Our proteomic data indicated that S. japonicum EVs included characteristic markers of exosomes, such as heat-shock proteins (HSPs 70 and 90), actin, elongation factor, and Rab proteins. Most proteins identified in the secretome of Leishmania, helminths, and other parasites were also observed in S. japonicum EVs, suggesting that exosome-like vesicles could play similar roles in protozoa and helminths 19,[26][27][28] . We found that some proteins involved in vesicle biogenesis, such as heat-shock proteins (HSPs 70 and 90) and members of the Rab GTPase family (Rab10, Rab11, and Rab8), were also detected in S. japonicum EVs. Rab proteins are essential regulators of intracellular vesicle transport between different subcellular compartments via processes including vesicle budding, mobility along the cytoskeleton, and membrane tethering and fusion 29 . These results highlight the hypothesis that extracellular vesicle formation is a highly conserved mechanism in eukaryotes 30 and could constitute an important mechanism for protein export in schistosomes.
Functional categorization of the identified EV proteins revealed a high proportion of nucleic acid-binding proteins, consistent with proteins identified in Trypanosoma cruzi and Leishmania secretomes 26,27 . An important inclusion to this category of proteins was RNA-binding proteins, including eukaryotic translation factors that have been assigned functions in both initiation and elongation and are known to play essential roles in gene expression regulation, modulating mRNA turnover, nucleocytoplasmic transport, and transcription 31 . Among these, eukaryotic translation elongation factors 1 alpha (eEF1A), are not only translation factors but also pleiotropic proteins highly expressed in tumors 32 . Additionally, data suggest that eEF1A proteins could not only activate the phospholipid and Akt signaling pathways that favor cell survival, but also block apoptosis and promote viral replication 33 . Thus, the release of such proteins suggests a regulatory role for S. japonicum EVs in parasite-host interaction and S. japonicum evasion from the host immune system.
Bioinformatics analyses indicated that exosome proteins could be involved in many pathways, including several already described in other parasites, such as the ubiquitin-proteasome system 34 . Among eukaryotes, the turnover of intracellular proteins is primarily mediated by the ubiquitin-proteasome system 35 , which is highly conserved from yeast to humans. Proteasomes are important for the survival and development of schistosomes 36,37 . In this study, the enrichment of proteasomes in S. japonicum EVs suggested that the ubiquitin-proteasome system could play an important regulatory role during schistosome infection. Proteasomal targeting could be a candidate strategy for anti-schistosome therapy due to the indispensable role of proteasomes in parasite invasion. We also identified several schistosome surface antigens in the S. japonicum EVs, such as a 22.6 kDa tegumental antigen (AAC67308), tegument antigen (I(H)A)(CAX71406), and major egg antigen (p40, CAX78232), which have been implicated in schistosome evasion of host immune responses 38,39 . Indeed, the parasite tegument covers the surface of the worms, constitutes a major interface between the parasite and its host, and plays a critical role in host-parasite interactions 40,41 . Parasite molecules expressed at the tegument surface are potential targets for immune or drug intervention 41,42 . Similarly, tegumental proteins of schistosomes are also considered potential targets for schistosomiasis control 23,41,42 . We observed a high proportion of membrane-and tegument-associated proteins in S. japonicum EVs. In addition, significant immune reactivity against S. japonicum EVs was observed when applying sera from S. japonicum-infected rabbits. Our results provide a basis for the identification of potential target molecules for the development of vaccines and schistosomiasis biomarkers.
One of the mechanisms of exosomes mediating cellular communication is through miRNAs that can be transported by EVs and exert regulatory functions in recipient cells 43,44 . We demonstrated that mammalian cells could internalize S. japonicum EVs and their associated miRNAs. These results suggest that S. japonicum EVs potentially function as signal messengers that regulate host gene expression, which could facilitate schistosome parasitism. In Drosophila, Bantam miRNA has been shown to target a tumor-suppress pathway, leading to cellular growth and the suppression of cellular death 45 . In the present study, we found an enriched Bantam in S. japonicum EVs, which can be transferred to liver cells via S. japonicum EVs. Consequently, we hypothesized that schistosome-specific miRNAs, such as Bantam, may be involved in the hepatic pathogenesis of schistosomiasis. In support of this, we determined the mRNA expression of three potential target genes (Gins4, Tysnd1, and Utp3) of schistosome Bantam miRNA in mice. Both in vivo animal studies and in vitro cell culture study clearly indicated a common down-regulation of mRNA expressions in the livers of S. japonicum infected mice (28 dpi and 35 dpi) and in liver cells treated with S. japonicum EVs. In addition, we observed that a considerable number of small RNAs isolated from S. japonicum EVs (1,284,966 reads, 3.43%) specifically mapped to the rabbit genome, including a known miRNA (ocu-miR-191-5p) with a relatively high number of reads (4,548 reads); further qRT-PCR analysis indicated that ocu-miR-191-5p was also enriched in the RNAs isolated from S. japonicum EVs. These results suggest that S. japonicum EVs could be important regulators of host-pathogen interactions.

Conclusion
We characterized the biochemical components of S. japonicum EVs using a proteomics approach and determined their associated small RNA populations using deep sequencing. We demonstrated that S. japonicum EVs, which contain parasite antigens, miRNAs, and potential virulence factors, can be internalized by host cells and transfer cargo miRNAs to recipient cells. These findings indicate that S. japonicum EVs may play an important regulatory Parasite cultures and cultured medium collection. New Zealand rabbits were percutaneously infected with approximately 1,500 S. japonicum cercariae (Anhui isolate, China). Schistosomes at the liver stage were collected from rabbits infected with S. japonicum at 28 dpi. Parasites were thoroughly and gently washed three times with 50 mL PBS (pH 7.4) and then maintained in preheated RPMI-1640 culture medium (HyClone, Logan, UT) containing 100 U of penicillin and 100 mg/mL of streptomycin (Sigma, St. Louis, MO, USA) at 37 °C under 5% CO 2 at a density of ~5 worm pairs /mL for 2 h. Following 2 h incubation, worms were microscopically examined to ensure their teguments were intact. Then, parasites and pellets were removed by centrifugation at 2,000 × g and 14,000 × g for 30 min each at 4 °C, respectively. The culture medium was collected, dialyzed in PBS for 24 h at 4 °C, and concentrated by centrifugal ultrafiltration through a 3 K NMWL membrane (Merck Millipore, Darmstadt, Germany). The supernatant was then filtered using a 0.22 μ m syringe filter (Merck Millipore) and transferred to a 15 mL polyallomer tube for further exosome isolation using a total exosome isolation kit as described below. In  addition, proteins from the culture medium without exosome isolation were directly precipitated by adding five volumes of cold acetone; the mixture was stored at − 20 °C overnight.

Isolation of exosome-like vesicles.
A total exosome isolation kit (Life Technologies, Carlsbad, CA, USA) was used for exosome isolation according to the manufacturer's instructions with minor modifications. All procedures were performed at 4 °C. In brief, the conditioned medium treated as described above was further centrifuged at 14,000 × g for 30 min and resulting pellets were discarded. Next, 0.5 volumes of the total exosome isolation reagent (Life Technologies) were added to the supernatants and incubated at 4 °C overnight. The supernatant from the final step was then centrifuged at 14,000 × g for 1 h. In parallel, the supernatant (residuum collected after exosome isolation) was collected. The resulting pellet was resuspended in 25 μ L PBS and stored at − 80 °C until further analysis.
Transmission electron microscopy. For electron microscopy, purified EVs were added to 200 mesh formvar-coated grids (Agar Scientific, Essex, UK) and allowed to dry at room temperature. The grids were washed with water and stained with 1% uranyl acetate (System Biosciences, Mountain View, CA, USA) for 5 min. After staining, the grids were washed once in 70% ethanol followed by four washes with molecular grade water. The grids were then loaded onto the sample holder of the transmission electron microscope (Hitachi H-7600, Tokyo, Japan) and exposed to an 80 kV electron beam for image capture.

Analysis of exosome-like vesicles by SDS-PAGE.
The concentrations of purified exosomes were determined using Bradford protein assays (Sangon Biotech, Shanghai, China). Proteins (5 μ g) from isolated S. japonicum EVs were separated using precast 4-20% polyacrylamide linear gradient gels (Bio-Rad, Hercules, CA, USA). A pre-stained protein standard (Thermo Scientific, Waltham, MA, USA) was used to track protein migration. After running, gels were stained by silver as previously described 46 and scanned using a Bio-Rad Molecular Imager FX system (Bio-Rad).
Enzymatic digestion of protein. The   14,000× g, filters were washed three times with 100 μ L UA buffer and then 100 μ L of dissolution buffer (50 mM triethylammonium bicarbonate, pH 8.5) twice. The tryptic peptides resulting from the digestion were extracted with 0.1% formic acid in 60% acetonitrile. The extracts were pooled and completely dried using a vacuum centrifuge.
In addition, nine of the gel blocks were excised and each slice was divided into 3 mm sections. Gel slices were destained using 30 mM potassium ferricyanide/100 mM sodium thiosulfate (1:1 v/v) and washed with Milli-Q water. The spots were incubated in 0.2 M NH 4 HCO 3 for 20 min and then lyophilized. The in-gel proteins were reduced with DTT (10 mM DTT/ 100 mM NH 4 HCO 3 ) for 30 min at 56 °C, then alkylated with IAA (50 mM IAA/100 Mm NH 4 HCO 3 ) in the dark at room temperature for 30 min. Gel pieces were briefly rinsed with 100 mM NH 4 HCO 3 and ACN, respectively. Finally, the protein suspension was digested with 2 μ g trypsin (Promega, Madison, USA) in 40 μ L 25 mM NH 4 HCO 3 overnight at 37 °C. Peptides were extracted as described above.
Mass spectrometry. For total exosomal protein identification, liquid chromatography/mass spectroscopy (LC-MS/MS, Thermo Scientific) analysis was performed using a Q Exactive mass spectrometer coupled to an Easy nLC system (Thermo Scientific). Trypsin-digested peptides (~5 μ g) were trapped and desalted on Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) and separated on a C18-reversed phase column (0.15 mm × 150 mm, Column Technology Inc. Fremont, CA). The Easy nLC system (Thermo Scientific) was used to deliver mobile phases A (0.1% formic acid in HPLC-grade water) and B (0.1% Formic acid in 84% acetonitrile) with a linear gradient of 4-50% B (50 min), 50-100% B (4 min), and then 100% B (6 min) at a flow rate of 250 nL/min. To acquire the MS data, a data-dependent top ten method was used, in which the ten most abundant precursor ions were selected for HCD fragmentation. For survey scans (m/z 300-1800), the target value was determined based on predictive Automatic Gain Control at a resolution of 70,000 at m/z 200 and dynamic exclusion duration of 25 s. Resolution for HCD spectra was set to 17,500 at m/z 200. Normalized collision energy was 27 eV and the under fill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%.
For in gel protein identification, the Ettan TM MDLC controlled by UNICORN TM software (GE Healthcare), a system for automated multi-dimensional chromatography was used for desalting and separation of peptides prior to on-line LTQ Velos (Thermo Scientific) analyses. In this system, peptide mixtures were desalted on RP trap columns (Zorbax 300 SB C18, Agilent Technologies), and then separated on a C18-reversed phase column (0.15 mm × 150 mm, Column Technology Inc. Fremont, CA). Mobile phase A (0.1% formic acid in HPLC-grade water) and mobile phase B (0.1% formic acid in 84% acetonitrile) were selected. Tryptic peptide mixtures were loaded onto the columns, and separation was done at a flow rate of 2 μ L/min by using the linear gradient buffer B described above. LTQ Velos (Thermo Scientific) equipped with a micro-spray interface was connected to the

Data analysis.
For total exosomal protein identification, data interpretation and protein identification were performed with the MS/MS spectra data sets using the Mascot search algorithm (v 2.2, Matrix Science) against the UniProtKB Schistosoma database (download at May 11, 2015, with 16,273 entries). The search parameters were trypsin enzyme, two missed cleavages, fixed modifications of carbamidomethyl, variable modifications of oxidation, a fragment ion mass tolerance of 0.10 Da, and peptide tolerance of 20 ppm. Only proteins with at least two peptide (filter by ion score ≥ 20 and false discovery rate < 0.01) uniquely assigned to the respective sequence were considered identified.
For in gel protein identification, MS/MS spectra were automatically searched against the Schistosoma database (described above) using the BioworksBrowser rev. 3.3 (Thermo Electron, San Jose, CA, USA). Protein identification results were extracted from SEQUEST out files with BuildSummary. Peptides were constrained to be tryptic and up to two missed cleavages were allowed. Carbamidomethylation of cysteines was treated as a fixed modification, whereas oxidation of methionine residues was considered as variable modifications. The mass tolerance allowed for the precursor ions was 2.0 Da and fragment ions was 0.8 Da. The protein identification criteria were based on Delta CN (≥ 0.1) and cross-correlation scores (Xcorr, one charge ≥ 1.9, two charges ≥ 2.2, three charges Gene ontology analysis. To obtain the molecular function, protein class, biological process, and pathway of the proteins involved, we searched the consortium databases of the protein classification software PANTHER (http://www.pantherdb.org/) using Mus musculus proteins as a reference.

Small RNA library preparation and bioinformatics analysis.
For the analysis of small RNAs, total RNA was extracted from S. japonicum secreted EVs using Trizol LS reagent (Life Technologies), and RNA quality was analyzed using an Agilent 2100 system (Agilent Technologies). Next, RNA was size-selected using 15% denaturing PAGE and libraries were prepared from the 18-30 nt fraction and ligated first to a 5′ RNA adaptor and then to a 3′ RNA adaptor using an Illumina small RNA Preparation Kit (version 1, Illumina). The small RNA libraries were subjected to sequencing using Illumina 50 bp single end sequencing performed on an Illumina Hiseq 2000 machine at the BGI (Beijing Genomics Institute, Shenzhen, China). The raw sequencing data were deposited with the NCBI SRA under project number PRJN305851.
By comparing our sequences with those in databases and selecting the genome location overlap between our data and the databases, sequenced small RNAs were annotated to different categories, including rRNA, tRNA, small nuclear RNA (snRNA), miRNAs, and small nucleolar RNA (snoRNA), by matching against sequences of noncoding RNAs collected in Rfam (Version 11.0) and the NCBI GenBank database (http://www.ncbi.nlm.nih. gov/) using BLAST. For miRNA analysis, these unmatched small RNAs were further analyzed against miRbase (version 21) and GenBank to determine known miRNAs or homologous miRNAs. Finally, unannotated small RNAs were subject to analysis for novel miRNA identification using the Mireap (http://sourceforge.net/projects/ mireap). RNAfold was used to predict hairpin-like structures.
qRT-PCR analysis of S. japonicum EV associated miRNAs. A stem-loop based qRT-PCR was used to validate the presence of miRNAs in S. japonicum EVs. Small RNAs isolated from S. japonicum EVs, culture medium, and residuum supernatants (collected after EVs isolation) were extracted using a mirVana PARIS Kit (Life Technologies) according to the manufacturer's instructions. In parallel, Sja-miR-1175, which was not found in the S. japonicum EV library was used as a negative control. The qRT-PCR analysis was performed as previously described 24 . Briefly, a stem-loop RT primer was used to reverse-transcribe mature miRNAs to cDNAs: (ocu-miR-191, GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACC AGC TG; Sja-miR-1175, GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACC AGT TG) by using a PrimeScript TM RT Reagent Kit (Takara) according to the manufacturer's instructions. PCR was performed in a Mastercycler ep realplex (Eppendorf). The 20 μ L PCR reaction included 2 μ L of RT product (1:3 dilution), 1 × SYBR Premix Ex Taq II (Takara), 0.5 μ M specific forward primer (ocu-miR-191_F, ATC GTA CGT GGG CAA CGG AAT C; miR-1175_F, ATC GTA CGT GGG TGA GAT TCA), 0.5 μ M common reverse primer (GCA GGG TCC GAG GTA TTC). The abundance of nicotinamide adenine dinucleotide dehydrogenase (NADH) (forward primer: CGA GGA CCT AAC AGC AGA GG; reverse primer: TCC GAA CGA ACT TTG AAT CC) was used as the internal control for normalization. The 2 −ΔCt method was used to calculate relative miRNA abundance. All reactions were performed in triplicate.

Analysis of antibody responses to S. japonicum EVs. Antibody responses to S. japonicum secreted
EVs in sera of rabbits (healthy and S. japonicum-infected) were evaluated using indirect ELISA and Western blot. Sera from rabbits infected with S. japonicum at 45 dpi were collected and pooled (n = 10). An ELISA method was performed as previously described 47 . The S. japonicum secreted EVs were coated as antigens on the ELISA plate with 1 μ g (100 μ L) per well of coating buffer (0.05 M sodium carbonate solution, pH 9.6) and kept at 4 °C overnight. To start the assay, plates were blocked with 5% bovine serum albumin in PBS for 1 h at 25 °C. After the plates were washed three times with PBST, diluted rabbit sera at different dilutions (1:10 and 1:50) in PBS containing 1% bovine serum albumin were added, incubated at 37 °C for 1 h, and washed with 1% Tween in PBS. An HRP-labeled goat anti-rabbit IgG secondary antibody (Kexin Bioscience, China) diluted 1:10,000 was added and incubated at 37 °C for 1 h. Next, the color reaction was developed by adding 3,3′ ,5,5′ -tetramethylbenzidine (TMB) substrate (BiYunTian Biotechnology Research Institute, Nantong, Jiangsu, China) to each well; 50 μ L/well of 2 M H 2 SO 4 was used to stop the reaction. Finally, the optical density (OD) in each well was measured at 450 nm using a Tecan Infinite M200 Pro plate reader (Tecan Group Ltd., Männedorf, Switzerland). The OD 450 values of sample wells above 2.1 times that of the negative control wells were considered positive 47 .
To obtain enough protein for Western blot (30 μ g), EVs were pooled from individual harvests (n = 4). The culture media and EV pellets containing 30 μ g of protein were lysed with Laemmli sample buffer and run on 10% gels. S. japonicum and 293 T cell lysates were used as controls. Proteins were transferred onto a PVDF membrane (Whatman International Ltd., Kent, UK). Membranes were blocked with 5% (w/v) skim milk powder in PBST and probed with primary rabbit serum (1:100, pooled from at least five rabbits at 45 dpi) overnight at 4 °C in 1% milk in PBST, followed by incubation with HRP-conjugated goat anti-rabbit-IgG secondary antibodies (Kexin Bioscience, Shenzhen, Guangzhou, China) at a 1:2000 dilution in 1% milk in PBST for 1 h at room temperature. After extensive washing, the blots were incubated for 1 min at room temperature with Immobilon Western