Characteristics and immunoprotective functions of three cysteine proteases from .

INTRODUCTION

Cysteine proteases from , including various proteins, are essential for its pathogenicity and serve as potential vaccine candidates. This study assesses the protective effects of three cysteine proteases (CsCP1-3).

METHODS

Mice immunized with recombinant CsCP1-3 and adjuvants were subsequently infected with metacercariae after three immunization rounds. Liver damage was evaluated through hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemical analyses. The levels of IgG1, IgG2a antibodies, and cytokines (IFN-g, IL-2, IL-4, and IL-10) were quantified by enzyme-linked immunosorbent assay (ELISA).

RESULTS

RT-qPCR revealed that CsCP1-2 exhibited the highest expression in newly encysted larvae (NEL), while CsCP3 was predominantly expressed in adult stages. Immunohistochemical localization confirmed that CsCP1-3 are present in the eggshells, syncytial layers of metacercariae, NEL cuticle, and adult intestines. Histological and immunohistochemical analysis demonstrated that the rCsCP1-3-immunized group displayed reduced liver inflammation and biliary fibrosis compared to the control group. The rCsCP1-3 induced a progressive increase in specific IgG1 and IgG2a antibody titers by the second week post-immunization. In the CsCP1-2 group, cytokines IFN-g, IL-2, IL-4, and IL-10 were elevated relative to the control, with particularly high levels of IFN-g and IL-10 in CsCP1, indicating a strong mixed Th1/Th2 immune response. In contrast, the CsCP3 immunization group exhibited a transient increase in cytokines (IFN-g, IL-2, IL-4, and IL-10) three days postinfection, which subsided after one to two weeks.

DISCUSSION

These findings suggest that CsCP1-3 elicit robust antibody and cellular immune responses, mitigating liver damage caused by infection. CsCP1, in particular, induces a potent mixed Th1/Th2 response, positioning it as a promising vaccine candidate.