Abstract
Human clonorchiasis has been increasingly prevalent in recent years and results in a threat to the public health in epidemic regions, motivating current strategies of vaccines to combat Clonorchis sinensis (C. sinensis). In this study, we identified C. sinensis paramyosin (CsPmy) from the cyst wall proteins of metacercariae by proteomic approaches and characterized the expressed recombinant pET-26b-CsPmy protein (101 kDa). Bioinformatics analysis indicated that full-length sequences of paramyosin are conserved in helminthes and numerous B-cell/T-cell epitopes were predicted in amino acid sequence of CsPmy. Western blot analysis showed that CsPmy was expressed at four life stages of C. sinensis, both cyst wall proteins and soluble tegumental components could be probed by anti-CsPmy serum. Moreover, immunolocalization results revealed that CsPmy was specifically localized at cyst wall and excretory bladder of metacercaria, as well as the tegument, oral sucker and vitellarium of adult worm. Both immunoblot and immunolocalization results demonstrated that CsPmy was highly expressed at the stage of adult worm, metacercariae and cercaria, which could be supported by real-time PCR analysis. Both recombinant protein and nucleic acid of CsPmy showed strong immunogenicity in rats and induced combined Th1/Th2 immune responses, which were reflected by continuous high level of antibody titers and increased level of IgG1/IgG2a subtypes in serum. In vaccine trials, comparing with control groups, both CsPmy protein and DNA vaccine exhibited protective effect with significant worm reduction rate of 54.3% (p<0.05) and 36.1% (p<0.05), respectively. In consistence with immune responses in sera, elevated level of cytokines IFN-γ and IL-4 in splenocytes suggested that CsPmy could induce combined cellular immunity and humoral immunity in host. Taken together, CsPmy could be a promising vaccine candidate in the prevention of C. sinensis regarding its high immunogenicity and surface localization.
Highlights
Human clonorchiasis, caused by the liver fluke Clonorchis sinensis (C. sinensis), has been increasingly prevalent in recent years, resulted from greater consumption of raw freshwater fish containing infective C. sinensis metacercariae [1]
The impact on public health of food-borne clonorchiasis is considerable since more than 35 million people are infected with C. sinensis and 601 million are at the risk of this neglected food-borne disease, which has been highly taken into account in regarding its serious complications [29]
The present study explored the potential role of C. sinensis paramyosin as a vaccine candidate, which was identified from the cyst wall of C. sinensis metacercariae by HPLC-MS/MS
Summary
Human clonorchiasis, caused by the liver fluke Clonorchis sinensis (C. sinensis), has been increasingly prevalent in recent years, resulted from greater consumption of raw freshwater fish containing infective C. sinensis metacercariae [1]. Including 15 million afflicted people in China, more than 35 million people globally were infected by this food-borne parasite [2,3]. Chronic infection by the carcinogenic parasite has been regarded to be responsible for other hepatobiliary diseases such as pyogenic cholangitis, cholelithiasis, cholecystitis and hepatic fibrosis [7]. Increasing infection of C. sinensis has led to negative socio-economic impact in epidemic regions and resulted in a threat to the public health. The complicated molecular mechanism involved in liver flukeassociated hepatobiliary diseases remains to be elucidated, motivating current strategies of vaccines to combat C. sinensis [8]
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