Optimization of in vitro Assays to Study the Efficacy of Anti-Leptospira Antibodies

Leptospirosis is a reemerging global zoonotic disease caused by Leptospira. Antibody-mediated protective response is commonly evaluated using animal models. Previously demonstrated in vitro growth inhibition test lacks consistent procedures. Thus, considering the 3Rs of animal research, it is ideal to develop an in vitro assay for the initial evaluation of antibodies that can potentially protect the animals before moving to the in vivo models. The objective of this study was to optimize in vitro assays to conduct a preliminary evaluation of antibody efficacy. First, we attempted to optimize the Leptospira growth inhibition assay (LGIA) in which L. interrogans serovar Manilae culture containing 10^3 to 10^7 bacteria was treated with polyclonal antiserum developed against this strain. Cultures were examined for the presence of Leptospira growth for 28 days. We observed that 10^4 bacteria treated with a 1:10 dilution of antiserum, for a 14-day incubation, would be an optimal combination for conducting this assay, and these conditions effectively inhibit the growth of Leptospira in the presence of antiserum. Moreover, another key mechanism for preventing bacterial growth is phagocytosis. As previous studies showed inconsistent findings on the phagocytosis of virulent strains, we attempted to evaluate the parameters for detecting intracellular Leptospira within THP-1 (a human monocytic cell line), which would further aid in optimizing the macrophage-infection model for assessing antibody efficacy in the antibody-mediated bacterial phagocytosis process. Upon THP-1 cell infection with Leptospira, we noted that CFSE (Carboxyfluorescein succinimidyl ester) labeling for bacteria was more effective in detecting intracellular Leptospira, compared to FITC (Fluorescein isothiocyanate)-conjugated polyclonal anti-Leptospira antibody, as FITC-conjugated antibody exhibited excessive fluorescence and non-specific binding to THP-1 cells. Next, after treating the infected THP-1 cells with gentamicin, we observed that treatment with gentamicin did not eliminate extracellular Leptospira from the cell culture. Further evaluation of this experiment is necessary, as our observations were invalid, as it was unclear whether the bacteria resided inside or outside of the cell. We concluded that LGIA is an appropriate testing strategy for evaluating antibody efficacy and can be optimized for various Leptospira species and conditions. However, in our experience, the macrophage model can be labor-intensive and not straightforward and may not be suitable for this purpose.