Fig 1.
CD5 and CD6 ectodomains bind PSC tegumental antigens.
(A) Biotin-labeled rshCD5, rshCD6 and BSA protein solutions (20 μg/mL) were sequentially incubated (4 times) with PSC suspensions (5,000 PSC; viability ≥90%), and pellets and solutions run on SDS-PAGE and further Western blotted with HRP-streptavidin. Lanes 1 and 2: protein solutions before (0x) and after 4 sequential incubations with PSC (4x), respectively. Lanes 3 to 7: PSC pellets after 0x to 4x sequential incubations. (B) ELISA assays showing the binding of increasing amounts of biotinylated rshCD5, rshCD6 and BSA proteins to PSEx-coated plates. (C) ELISA assays showing the binding of supernatants from sequential PSC incubations of biotin-labeled rshCD5, rshCD6 and BSA depicted in (A), to PSEx-coated plates. N.D. not detected. (*) Significant differences (Student’s t-test, P <0.05) respect to 0x results.
Fig 2.
Tegumental antigens recognized by rshCD5 and rshCD6 are different.
(A) ELISA assays showing binding of rshCD5 and rshCD6 to PSEx-coated plates undergoing metaperiodate treatment (NaIO4) or not (control). Binding to metaperiodate-resistant antigens was assessed as the percentage of absorbance values in treated wells respect to untreated wells (triplicates in each case). (B) Competition binding assays in which binding of a fixed amount of biotinylated rshCD5 (20 μg/mL; left) or rshCD6 (10 μg/mL; right) to PSEx-coated ELISA were competed with increasing amounts of unlabeled rshCD6 and rshCD5 proteins, respectively. Ligand overlapping was assessed (in triplicates) as the percentage of absorbance values in competed wells respect to non-competed wells (0 μg/mL of unlabeled protein). (C) 2D SDS-PAGE resolved PSEx fractions were either silver stained (left) or electro-transferred onto PVDF membranes for further Western blotting with biotin-labeled rshCD5 (middle) or rshCD6 (right). (*) Significant differences (Student’s t-test, P <0.05) respect to control (A) or uncompleted (B) wells.
Table 1.
Potential parasite ligands for rshCD5 and rshCD6 identified by 2D SDS-PAGE and further MALDI-TOF/TOF mass spectrometry analysis.
Fig 3.
Native membrane-bound CD5 and CD6 receptors retain PSEx-binding capacity.
(A) Flow cytometry analyses of 2G5, 2G5-CD5.wt or 2G5-CD6.wt cells stained with increasing amounts of FITC-labeled PSEx. Represented are the mean fluorescence intensity (MFI) values (left) and a representative flow cytometry histogram from each case (right). (B) Competition binding experiments in which 2G5-CD5.wt or 2G5-CD6.wt cells were stained with a fixed suboptimal amount of FITC-labeled PSEx in the presence or absence of different amounts of unlabeled rshCD5 or rshCD6 proteins. Both experiments were performed in quadruplicates and results are shown as mean +/- SD. (*) Significant differences (Student’s t-test, P <0.05) respect to 2G5 cells (A) or cells with 0 μg of competing proteins (B).
Fig 4.
PSEx-induced cytokine production by naïve cells from CD5-/- and CD6-/- mice.
(A) Peritoneal cells from naïve CD5-/- (n = 3, pooled) and wild-type (n = 3, pooled) control mice, were cultured for 72 h in the presence of increasing amounts of PSEx, and then cytokine production in supernatants was analyzed by capture ELISA. Results are displayed as fold increases respect to unstimulated cells (mean +/- SEM). (B) Same as in (A) for CD6-/- and wild-type control mice. Both experiments were performed in quadruplicates. (*) Significant differences (Student’s t-test, P <0.05) respect to unstimulated cells.
Fig 5.
Modulation of PSEx-induced cytokine production by rshCD5 or rshCD6.
(A) Peritoneal cells from naïve wild-type C57BL/6 mice (n = 3, pooled) were stimulated with a fixed concentration of PSEx (20 μg/mL) for 72 h in the presence of the indicated amounts of rshCD5, BSA or LPS. Results are displayed as cytokine concentration in supernatants (mean +/- SEM). (B) Same as in (A) using the indicated amounts of rshCD6 instead of rshCD5. Both experiments were performed in quadruplicates. (*) Significant differences (Student’s t-test, P <0.05) respect to cells stimulated only with PSEx.
Fig 6.
Infusion rshCD5 or rshCD6 protects mice from secondary CE.
The anti-parasite prophylactic potential of rshCD5 or rshCD6 was assessed in the murine model of secondary CE by infusing PBS, rshCD5, rshCD6 or BSA (25μg/dose/mouse, i.p.) 1 h before and 1 h after i.p. inoculation of 2,000 highly viable PSC/mouse into Balb/c mice (n = 6–8 mice per experimental group). At 14 months post infection, mice were euthanized, and the peritoneal hydatid cysts within each mouse were counted and weighted. Results are displayed as percentage of infected mice (A), number (mean +/- SEM) of hydatid cysts per mouse (B), and total wet weight (mean +/- SEM) of hydatid cysts per mouse (C). The experiment was performed twice. (*) Significant differences (P <0.05) respect to the PBS group were assessed by either Fisher’s exact test (A) or Mann-Whitney U-test (B and C).