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

## TL;DR

This study develops a new diagnostic test for leptospirosis by detecting specific disease-causing proteins in the blood of infected animals, offering hope for improved diagnosis and treatment.

## Contribution

The novel monoclonal antibody-based immunoassay detects virulence-modifying proteins in leptospirosis for the first time, advancing diagnostic and pathogenesis understanding.

## Key findings

- Monoclonal antibodies successfully detect VM proteins in the blood of infected hamsters.
- VM proteins are secreted during infection, providing evidence for their role in disease pathogenesis.
- High-affinity antibodies with sub-picomolar dissociation constants were developed for VM protein detection.

## Abstract

Leptospirosis, a globally 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 binding to homologous antigens, with
sub-picomolar dissociation constants (Kd = 1.41E-09 for 5F8 and
Kd <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.

This research addresses the global health issue of leptospirosis, a neglected
tropical disease that still lacks early and reliable diagnostic methods
despite being known for over a century. The study has developed a novel test
using specially designed antibodies to detect specific proteins related to
the disease in the blood of infected hamsters. These proteins are linked to
the pathogen's ability to cause illness. The successful detection of these
proteins in the bloodstream is a significant advancement, as it not only
improves our understanding of the disease's progression but also lays the
groundwork for developing new diagnostic tools. This could lead to earlier
and more accurate diagnoses of leptospirosis, potentially saving lives and
reducing the impact of the disease globally.