Background/Objectives: In the past, FhSAP-2, an 11.5 kDa recombinant protein belonging to the Fasciola hepatica saposin-like/NK-lysin family, has been shown to induce over 60% partial protection in immunized rabbits and mice when challenged with F. hepatica metacercariae. However, despite FhSAP-2 being a promising vaccine candidate, its hydrophobic nature has made its purification a challenging process. The present study aimed to determine whether a modified 9.8 kDa variant of protein (mFhSAP-2), lacking a string of 16 hydrophobic amino acids at the amino terminus and a dominant Th1 epitope, could retain its immunogenic and Th1-inducing properties. Methods: RAW264.7 cells were stimulated with mFhSAP-2, and TNFα levels were determined. C57BL/6 mice were immunized with mFhSAP-2 alone or emulsified with Montanide ISA50. Total anti-mFhSAP-2 IgG subtypes, along with their avidity and titers, were measured using ELISA. The T cell proliferation index and levels of CD4+/CD8+ and IFNγ/IL-4 ratios were determined. Results: In vitro, mFhSAP-2 induced dose-dependent TNFα production in RAW264.7 cells. In vivo, mice immunized with mFhSAP-2 or mFhSAP-2+ISA50 developed high-avidity IgG2a and IgG2c antibodies at levels that were significantly higher than IgG1 antibody levels. However, the mFhSAP-2+ISA50 formulation induced higher and more homogenous antibody titers than mFhSAP-2, suggesting that an adjuvant may be required to enhance mFhSAP-2 immunogenicity. Immunization with mFhSAP-2+ISA50 also induced significantly higher activated CD4+/CD8+ T cell ratios and IFNγ/IL-4 ratios compared to naïve mice. Conclusions: Our results demonstrate that mFhSAP-2 retained its immunogenicity and Th1-polarizing properties, which were enhanced by the Montanide ISA50 adjuvant. The present study highlights the feasibility of inducing Th1-associated immune responses in mice using mFhSAP-2 as an antigen. Further studies are required to assess the potential application of the mFhSAP-2+ISA50 formulation as a vaccine against F. hepatica in natural hosts such as cattle and sheep, which could contribute to improved control and aid in the prevention and eradication of F. hepatica infection.

Background/Objectives: FhSAP-2 is a 11.5kDa recombinant protein belonging to the Fasciola hepatica saposin-like/NK-lysin protein family. It has shown to induce partial protection >60% (reduction in parasite burden and parasite egg output) against F. hepatica challenge infection when delivered subcutaneously in rabbits and mice, either emulsified in Freund’s adjuvant or as inclusion bodies. The protection induced by FhSAP-2 was associated with increased levels of IgG2a and IFNγ. However, despite FhSAP-2 being a promising vaccine candidate, its hydrophobic character has made its purification a challenged process. The present study aimed to determine whether a modified 9.8kDa variant of protein (mFhSAP-2), lacking the string of 15 hydrophobic amino acids at amino terminus containing a dominant Th1-epitope, could retain its immunogenic and Th1-inducing properties. Methods: RAW264.7 cells were stimulated with mFhSAP-2, and levels of TNFα were determined. C57BL6 mice were immunized with mFhSAP-2 alone or emulsified with Montanide ISA50. Levels of each specific anti-mFhSAP-2 IgG subtypes, their avidity and titers were measured by ELISA. T-cell proliferation index as well as levels of CD4+/CD8+ and IFNγ /IL-4 ratios were determined. Results: In vitro, mFhSAP-2 induced dose-dependent TNFα production in RAW264.7 cells. In vivo, mice immunized with mFhSAP-2 alone induced 1.76-fold higher than IgG2a and 1.28-fold higher than IgG1, whereas mice immunized with mFhSAP-2+ISA50 developed 2.16-fold more IgG2c than IgG2a and 1.49-fold more IgG2c than IgG1. All mice developed high-avidity IgG2a and IgG2c antibodies. Mice immunized with mFhSAP-2+ISA50 developed higher IgG2a and IgG2c titers (1:6,400 and 1:25,600, respectively) than those immunized with mFhSAP-2 alone (1:1,600 and 1:3,200, respectively). Immunization with mFhSAP-2+ISA50 also induced significantly high activated CD4+/CD8+ T-cells and IFNγ/IL-4 ratios. Conclusions: Our results showed that mFhSAP-2 retained its immunogenicity and Th1-polarizing properties, which were enhanced by Montanide ISA-50 adjuvant.