Development of leptospiral virulence-modifying protein detection assay: implications for pathogenesis and diagnostic test development

Leptospirosis, a globally significant significant neglected tropical disease, continues to lack early and reliable diagnostic methods despite over a century since the discovery of the disease and its etiological agent. Previously, we identified the pathogen-specific paralogous PF07598 gene family encoding virulence-modifying (VM) exotoxins, which play a critical role in leptospirosis pathogenesis. In this study, we developed a monoclonal antibody (mAb)-based capture immunoassay that detects VM proteins in the blood of experimental hamster models, validating the hypothesis that VM proteins function as secretory exotoxins that mediate disease pathogenesis. Monoclonal antibodies were generated against a natural variant, LA0591, a VM protein containing a conserved C-terminal DNase toxin domain but lacking N-terminal ricin B-like lectin domains. Epitope mapping identified specific linear epitopes targeted by mAbs 5F8, 5G10, and 6A5, with distinct binding regions confirmed through binning and peptide mapping. These mAbs demonstrated high-affinity high-affinity binding to homologous antigens, with sub-picomolar dissociation constants (K_d = 1.41E-09 for 5F8 and K_d <1.0E-12 for 5G10 and 6A5) and cross-reacted with full-length recombinant VM proteins expressed in E. coli. Immunoblotting revealed increased expression of VM proteins by L. interrogans serovar Copenhageni strain L1-130 under in vivo-like conditions. Using mAbs 6A5 and 5F8, a capture ELISA detected circulating VM proteins in the serum and urine from infected hamsters, confirming the secretion of these proteins during infection. This study provides the first evidence of secreted leptospiral exotoxins in the bloodstream of infected animals, advancing the understanding of leptospirosis pathogenesis and establishing a basis for developing novel diagnostic approaches.