Role of T lymphocytes and papain enzymatic activity in the protection induced by the cysteine protease against Schistosoma mansoni in mice

Abstract

Papain, an experimental model protease, was used to decipher the protective mechanism(s) of the cysteine peptidase-based schistosomiasis vaccine. To examine the role of T lymphocytes, athymic nude (nu/nu) and immunocompetent haired (nu/+) mice were subcutaneously (sc) injected with 50 µg active papain two days before percutaneous exposure to 100 cercariae of Schistosoma mansoni. Highly significant (P < 0.005) reductions in worm burden required competent T lymphocytes, while significant increases (P < 0.05) of >80% in dead parasite ova in the small intestine were independent of T cell activity and likely relied on the innate immune axis. To investigate the role of enzymatic activity, immunocompetent mice were sc injected with 50 µg active or E-64-inactivated papain two days before exposure to cercariae. The reductions in worm burden were highly significant (P < 0.0001), reaching >65% and 40% in active and inactivated papain-treated mice, respectively. Similar highly significant (P < 0.0001) decreases of 85% in the viability of parasite ova in the small intestine occurred in both active and inactivated papain-treated mice. These findings indicated that immune responses elicited by one or more papain structural motifs are necessary and sufficient for induction of considerable parasite and egg attrition. Correlates of protection included IgG1-dominated antibody responses and increases in the levels of uric acid and arachidonic acid in the lung and liver upon parasite migration in these sites. Identification of the shared patterns or motifs in cysteine peptidases and evaluation of their immune protective potential will pave the way to the development of a safe, efficacious, storage-stable, and cost-effective schistosomiasis vaccine.

Keywords: Antibody response; Nude mice; Papain; Schistosoma mansoni; Uric and arachidonic acid; Vaccine.

Graphical abstract

Fig. 1

Diagrammatical representation of the experimental design. (A) Comparison of the effects of papain on S. mansoni infection in immunocompetent (nu/+) versus athymic (nu/nu) mice. (B) Evaluation of the effects of active and inactive papain on S. mansoni infection in immunocompetent (nu/+) mice. Each diagram represents two separate experiments.

Fig. 2

Haematoxylin-eosin-stained paraffin liver sections 40 days after S. mansoni infection. Immunocompetent (A, B) and athymic (C, D) mice were treated with 0 (A, C) or 50 (B, D) μg papain two days before percutaneous exposure to 100 cercariae. Each figure is representative of 5 mice per group. Magnification: 200x.

Fig. 3

Effect of pretreatment with active or inactivated papain on parasitological parameters in immunocompetent nu/+ mice. Mice (8 to 10 per group) were pretreated with 0 (controls, left column in every panel) or 50 μg active (papain-pretreated, middle column in every panel) or E-64-inactivated (inactivated papain-pretreated, right column in every panel) papain before infection with 200 cercariae of S. mansoni. Parasitological parameters in individual mice were assessed 43 days after infection. Reduction % = mean number in untreated control mice – mean number in papain- pretreated mice/mean number in untreated control mice × 100. Significance of differences between the 3 groups was assessed by ANOVA and found to be highly significant (P < 0.0001) for all parameters tested. Significance of differences between papain-treated and control mice, assessed using One-Way Anova with post test and Mann-Whitney test, are shown for every parameter (P). Reduction in worm burden and liver egg counts in mice pretreated with inactivated papain was significantly (P < 0.005) lower than in mice pretreated with active papain.

Fig. 4

Paraffin liver sections 49 days after S. mansoni infection. Immunocompetent mice were treated with 0 (left vertical panel, A, D, G) or 50 μg active (middle vertical panel, B, E, H) or E-64-inactivated (right vertical panel, C, F, I) papain two days before percutaneous exposure to 100 cercariae. Each image showing staining with haematoxylin-eosin (A–C), irrelevant rabbit IgG (D–F), or anti-uric acid (G–I) antibody is representative of 5 mice per group. Magnification: 200x.

Fig. 5

The serum antibody isotype response to SmCB1 is representative of two experiments. Serum samples from three to five untreated (Infection Control), and active (Papain Pretreated) and inactivated (Inactive Papain) papain-pretreated mice were tested PI for binding to SmCB1 in replicate ELISAs. The serum samples were diluted 1:500, 1:250 and 1:25 to detect total, IgG class, and IgA antibodies, respectively. The columns represent the mean delta absorbance (with the mean values of serum samples from 3 naïve mice subtracted), and the vertical bars denote the SD about the mean. Significant differences (Student's t-test) from control values at P < 0.05 are indicated by an asterisk (*). IgE antibodies were not detected in serum samples diluted 1:25.

Fig. 6

Each point represents the mean serum uric acid or lipid levels for five individual mice per group ± SD. Interval 0 represents the values recorded in naïve mice. Significant differences (One Way ANOVA and Student's t-test) at P < 0.05 are indicated by an asterisk (*).

Fig. 7

Immunohistochemical assays of lung uric acid and ARA. Lung sections 7 days PI were reacted with an anti-uric acid (A–D) or anti-ARA (E–H) antibody in repeated immunohistochemical assays. Lung sections of naïve (A, E) and infected control (B, F) mice were negative. Sections of lung of active (C, G) and inactivated (D, H) papain-treated mice showed moderate reactivity. The images shown are representative of the consistently recorded reactivity for each mouse group at the specified interval. Magnification: 200x.

Fig. 8

Immunohistochemical assays of liver uric acid and ARA. Liver sections were reacted with an anti-uric acid (A–C) or anti-ARA (D–F) antibody in repeated immunohistochemical assays, 17 (A, D), 24 (B, E) days PI, and at perfusion (C, F) Sections of naïve mice were consistently negative (not shown), in contrast to sections from infected (left figure in each panel), active papain-treated (middle figure in each panel), and inactivated papain-treated (right figure in each panel) mice. The images shown are representative of the consistently recorded reactivity for each mouse group in the two experiments at the specified interval. The arrows point to intragranulomar eggs. Magnification: 200x.