A Modified Variant of Fasciola hepatica mFhSAP-2 as a Recombinant Vaccine Candidate Induces High-Avidity IgG2c Antibodies and Enhances T Cell Activation in Immunized C57BL/6 Mice

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.