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• Recombinant chimeric form of the large extracellular loop of Ov-TSP-2 and O. viverrini leucine aminopeptidase, designated rOv-TSP-2-LAP, was produced in bacteria. • Hamsters immunized with recombinant rOv-TSP-2-LAP produced humoral and cellular immune responses. • Hamsters vaccinated with rOv-TSP-2-LAP had significantly reduced fluke burdens compared to control animals that received adjuvant alone. Opisthorchiasis is a serious public health problem in East Asia and Europe. The pathology involves hepatobiliary abnormalities such as cholangitis, choledocholithiasis and tissue fibrosis that can develop into cholangiocarcinoma. Prevention of infection is difficult as multiple social and behavioral factors are involved, thus, progress on a prophylactic vaccine against opisthorchiasis is urgently needed. Opisthorchis viverrini tetraspanin-2 (Ov -TSP-2) was previously described as a potential vaccine candidate conferring partial protection against O. viverrini infections in hamsters. In this study, we generated a recombinant chimeric form of the large extracellular loop of Ov -TSP-2 and O. viverrini leucine aminopeptidase, designated r Ov -TSP-2-LAP. Hamsters were vaccinated with 100 and 200 µg of r Ov -TSP-2-LAP formulated with alum-CpG adjuvant via intraperitoneal injection and evaluated the level of protection against O. viverrini infection. Our results demonstrated that the number of worms recovered from hamsters vaccinated with either 100 or 200 µg of r Ov -TSP-2-LAP were significantly reduced by 27% compared to the adjuvant control group. Furthermore, the average length of worms recovered from animals vaccinated with 200 μg of r Ov -TSP-2-LAP was significantly shorter than those from the control adjuvant group. Immunized hamsters showed significantly increased serum levels of anti-r Ov -TSP-2 IgG and IgG1 compared to adjuvant control group, suggesting that r Ov -TSP-2-LAP vaccination induces a mixed Th1/Th2 immune response in hamsters. Therefore, the development of a suitable vaccine against opisthorchiasis requires further work involving new vaccine technologies to improve immunogenicity and protective efficacy. Image, graphical abstract [ABSTRACT FROM AUTHOR]