Identification of a linear B-cell epitope on the Schistosoma japonicum saposin protein, SjS']jSAP4: Potential as a component of a multi-epitope diagnostic assay

Background Schistosoma japonicum is one of three major species of blood flukes causing schistosomiasis, a disease, which continues to be a major public health issue in the Philippines. SjSAP4, a member of a multigene family of saposin-like proteins, is a recognized immunodiagnostic biomarker for schistosomiasis japonica. This study aimed to identify linear B-cell epitopes on SjSAP4 and to validate their potential as components of a multi-epitope assay for the serological diagnosis of schistosomiasis japonica. Methodology SjSAP4-derived peptides were expressed as GST-peptide-fused proteins and these were Western blot probed with human serum samples from S. japonicum Kato-Katz (KK)-positive individuals and uninfected controls. A core epitope was further identified by Western blotting through probing a series of truncated peptides with the schistosomiasis patient sera. The diagnostic performance of the core epitope-containing peptides and the full-length SjSAP4 was evaluated by enzyme-linked immunosorbent assay (ELISA) using a panel of sera collected from subjects resident in a schistosomiasis-endemic area of the Philippines. Main findings As a result of the peptide mapping, one peptide (P15) was found to be highly immunogenic in the KK-positive individuals. We subsequently showed that -S163QCSLVGDIFVDKYLD178is a core B-cell epitope of P15. Subsequent ELISAs incorporating SjSAP4, SjSAP4-Peptide and SjSP-13V2-Peptide showed a sensitivity of 94.0%, 46.0% and 74.0%, respectively, and a specificity of 97.1%, 100% and 100%, respectively. Notably, complementary recognition of the B-cell epitopes (SjSAP4-Peptide and SjSP-13V2-Peptide) was observed in a subset of the KK-positive individuals. A dual epitope-ELISA (SjSAP4-Peptide + SjSP-13V2-Peptide-ELISA) showed a diagnostic sensitivity of 84.0% and a specificity of 100%. Conclusions/Significance In this study, -S163QCSLVGDIFVDKYLD178- was identified as a dominant linear B-cell epitope on SjSAP4. This peptide and the complementary recognition of other B-cell epitopes using sera from different KK-positive individuals can provide the basis of developing a multiepitope assay for the serological diagnosis of schistosomiasis. Author summary The recent road map (2021-2030) released by WHO for controlling or eliminating neglected tropical diseases (NTDs) highlights diagnostics as a major focus. Development and deployment of accurate, affordable and field-friendly diagnostics/surveillance tools will be crucial for the control and elimination of schistosomiasis. Multi-epitope chimeric antigens, constructed based on linear B-cell epitopes identified from highly antigenic antigens, may achieve not only an equivalent or superior diagnostic performance compared to the parent immunogens but also exhibit more optimal physicochemical properties. However, to date, only a limited number of linear B-cell epitopes have been identified for the serological diagnosis of schistosomiasis. In this study, we identified a linear B-cell epitope (-S163QCSLVGDIFVDKYLD 178-) on SjSAP4, a recognized immunodiagnostic biomarker for schistosomiasis japonica, and validated its potential as a component of a multi-epitope assay for the serological diagnosis of schistosomiasis japonica. Notably, differential recognition of B-cell epitopes (SjSAP4-Peptide and SjSP-13V2-Peptide) was observed in a subset of subjects positive by the Kato-Katz technique for the disease. A dual epitope-ELISA (SjSAP4-Peptide + SjSP-13V2-Peptide-ELISA) showed a superior diagnostic performance (84.0% sensitivity and 100% specificity) than individual-epitope ELISAs. The findings in this study provide support for the development of multi-epitope antigenbased diagnostics for schistosomiasis.