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cei4667-sup-0001-fS1.tif1532K

Fig. S1. Production of HaloTag, Halo-tagged C4beta and Halo-tagged ed1 as bacterial fusion proteins used covalently coupled to magnetic beads for competition enzyme-linked immunosorbent assay (ELISA). (a) Schematic representation of HaloTag fusion peptides generated. The HaloTag protein (boxed) is attached N-terminally to the sequences of interest via a 16 aa linker containing a tobacco etch virus (TEV) protease cleavage site. The six C-terminal amino acids of the linker sequence remaining attached to the peptides after cleavage are shown (underlined). HaloTag protein linked to Schistosoma mansoni tetraspanning orphan receptor (rSmTORed1) loop is denoted as HALOed1 and HaloTag fused to a 26 aa C4β chain stretch is termed HALOC4beta (human C4β chain peptide S206). Local sequence alignment over the C-terminal 27 aa was performed with ClustalW2 and then edited manually (identical amino acids in bold letters, similar amino acids shaded in grey). (b) Protein samples extracted from BL21 4·5 h after induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). HaloTag (HALO) and Halo-tagged (HALOC4 beta, HALOed1) peptides in crude Escherichia coli extracts visualized by Coomassie stain (left) and detected by Western blot probed with monoclonal anti-ed1 antibody AbDy04644·1 (right).

cei4667-sup-0002-fS2.tif199K

Fig. S2. Antibodies against recombinant first extracellular domain of Schistosoma mansoni tetraspanning orphan receptor (rSmTORed1) generated in mice do not recognize the homologous stretch on the complement C4 β chain. Total immunoglobulin (Ig) detection in sera of individual BALB/c mice immunized with rSmTORed1 (n = 3; mice 1·1–1·3) and a control mouse injected with phosphate-buffered saline (PBS) only (mouse 3·1). rSmTORed1 = purified histidine (HIS)-tagged SmTORed1; rSmTORed1/C4beta/HALO control tobacco etch virus (TEV) cut = SmTORed1, human C4β chain peptide S206 (C4beta) produced as HALO-tagged peptides or HALO-tag alone purified by TEV protease digestion.

cei4667-sup-0003-fs3.tif1018K

Fig. S3. Titration of sera from individual mice before and after immunization analysing anti-recombinant first extracellular domain of Schistosoma mansoni tetraspanning orphan receptor (rSmTORed1) immunoglobulin (Ig) (IgG, IgM, IgA) responses. Open and filled symbols denote Ig levels determined at days −7 and 35, respectively. Data points for the three mice (n = 3) of the strain indicated immunized with antigen and (a,b) complete Freund's adjuvant/incomplete Freund's adjuvant (CFA/IFA) or (c,d) muramyl dipeptide (MDP) adjuvant are shown in the left panel; values for the control mice (n = 3) immunized with adujvant/phosphate-buffered saline (PBS) only are shown in the right panel. The dilution determined for further screening of sera in the different settings is boxed. It was set according to optical density (OD) values at 450 nm still within but near the boarder of the plateau reached at low serum dilutions.

cei4667-sup-0004-fs4.tif710K

Fig. S4. Total immunoglobulin (Ig) amounts in sera of individual mice immunized with recombinant first extracellular domain of Schistosoma mansoni tetraspanning orphan receptor (rSmTORed1) and the control groups injected with inclusion bodies measured in sera pre-immunization and after the first boost. Ig levels in mice immunized with rSmTORed1 (antigen, ag; n = 10), inclusion bodies (i. bodies; n = 5) or PBS (n = 5) in complete Freund's adjuvant/incomplete Freund's adjuvant (CFA/IFA) were monitored at days −7 and 35 of the trial. Sera for the enzyme-linked immunosorbent assay (ELISA) assay were diluted as determined by end-point titrations for the different mouse strain adjuvant combinations that was (a) 1:6400 for C57BL/6 mice injected with antigen in CFA/IFA and (b,c) 1:3200 for BALB/c and C57BL/6 mice immunized with muramyl dipeptide (MDP). Statistical analyses used were: Student's t-test to compare titre at days −7 and 35 and one-way analysis of variance (anova) to compare titres at day 35; n.s.: statistically not significant; statistically significant *(P < 0·05), **(P < 0·01), ***(P < 0·001).

cei4667-sup-0005-fs5.tif558K

Fig. S5. Recombinant first extracellular domain of Schistosoma mansoni tetraspanning orphan receptor (rSmTORed1) complete Freund's adjuvant/incomplete Freund's adjuvant (CFA/IFA)-induced antibody response in BALB/c mice after challenge with Schistosoma mansoni infection. Immunoglobulin (Ig)M (a) and IgA (b) serology in mouse sera sampled at days −7, 35 and 107 (serum dilution both 1:1600). BALB/c mice were immunized with rSmTORed1 (antigen, ag) or control groups immunized with inclusion bodies (i. bodies) or buffer only [phosphate-buffered saline (PBS)] using complete CFA/IFA as adjuvant. All groups were infected with S. mansoni cercariae at day 55. Groups consisted of n = 10 animals. Arrows indicate injections at days 0 (prime), 21 (boost 1) and 42 (boost 2).

cei4667-sup-0006-fs6.tif292K

Fig. S6. The cytokine secretion profile of spleen cell cultures stimulated with antigen of immunized BALB/c mice was maintained after the infection challenge. Measurement of cytokines in the supernatant of spleen cell cultures after stimulation with recombinant first extracellular domain of Schistosoma mansoni tetraspanning orphan receptor (rSmTORed1) (a) 14 days after the second boost or (b) after immunization challenge experiment (trial 1) after 8 weeks (day 114) of infection. Number of spleens analysed: (a) n = 3/2 (rSmTORed1/PBS), (b) n = 2/2 (rSmTORed1/PBS). The bars represent the mean values ± standard deviation (s.d.) of either three or two (rSmTORed1/PBS) different samples each measured in triplicate. Splenocytes of all mice were confirmed to have the capacity for release of cytokines in response to stimulation with concanavalin A.

cei4667-sup-0007-fs7.tif152K

Fig. S7. Detection of anti-recombinant first extracellular domain of Schistosoma mansoni tetraspanning orphan receptor (rSmTORed1)-specific antibodies in sera from BALB/c mice (n = 5) immunized with radiation-attenuated cercariae. Total immunoglobulin (Ig) to rSmTORed1 was measured in pre-immune sera (day −7), after the first (day 35) and second boost (day 63). Results for the same sera diluted 1:3200 and 1:6400 are shown.

cei4667-sup-0008-si.doc200K

Table S1. Primer list used for cloning of Halo-tagged fusion constructs.

Appendix S1. Supplementary materials and methods.

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