Characterization of Spirometra erinaceieuropaei Plerocercoid Cysteine Protease and Potential Application for Serodiagnosis of Sparganosis

Background Sparganosis is a neglected but important food-borne parasitic zoonosis. Clinical diagnosis of sparganosis is difficult because there are no specific manifestations. ELISA using plerocercoid crude or excretory–secretory (ES) antigens has high sensitivity but has cross-reactions with other helminthiases. The aim of this study was to characterize Spirometra erinaceieuropaei cysteine protease (SeCP) and to evaluate its potential application for serodiagnosis of sparganosis. Methodology/Principal Findings The full length SeCP gene was cloned, and recombinant SeCP (rSeCP) was expressed and purified. Western blotting showed that rSeCP was recognized by the serum of sparganum-infected mice, and anti-rSeCP serum recognized the native SeCP protein of plerocercoid crude or ES antigens. Expression of SeCP was observed at plerocercoid stages but not at the adult and egg stages. Immunolocalization identified SeCP in plerocercoid tegument and parenchymal tissue. The rSeCP had CP activity, and the optimum pH and temperature were 5.5 and 37°C, respectively. Enzymatic activity was significantly inhibited by E-64. rSeCP functions to degrade different proteins and the function was inhibited by anti-rSeCP serum and E-64. Immunization of mice with rSeCP induced Th2-predominant immune responses and anti-rSeCP antibodies had the potential capabilities to kill plerocercoids in an ADCC assay. The sensitivity of rSeCP-ELISA and ES antigen ELISA was 100% when performed on sera of patients with sparganosis. The specificity of rSeCP-ELISA and ES antigen ELISA was 98.22% (166/169) and 87.57% (148/169), respectively (P
Author Summary Sparganosis is a neglected tropical disease; its diagnosis is difficult and it is often misdiagnosed. ELISA using the crude or ES antigens of plerocercoids cross reacts with other helminthiases. Cysteine protease is a type of hydrolase and plays important roles in the development and survival of parasites; it has been used for diagnostic markers and vaccine targets for some parasitic diseases. In this study, a 36 kDa Spirometra erinaceieuropaei cysteine protease (SeCP) was expressed and purified. The results showed that SeCP was a plerocercoid stage-specific protein located in the teguments and parenchymal tissue. The rSeCP had cysteine protease activity and functioned to degrade host proteins. Vaccination of mice with rSeCP induced high levels of IgG1 and anti-rSeCP antibodies with the ability to kill plerocercoids in an ADCC assay. The rSeCP had a high sensitivity and specificity for detecting anti-plerocercoid antibodies, and could be used as a potential antigen for serodiagnosis of sparganosis.