Fig. 2: Blocking activity of polyclonal immune serum IM1S significantly reduces in vitro infection by exerting a lytic effect on trypomastigotes.

a Trypomastigote-blocking effects on COLO-N680 cell infection assessed using sera from three mice immunised with TcPOP. Antisera were added at three concentrations and compared with naïve serum. Infection reduction is presented as the percentage of infected cells 72 h post-infection with trypomastigotes exposed to antisera for 4 h (Fig. 1d). Data are presented as mean ± SD from three independent biological replicates with two technical replicates per group, and statistical significance was assessed using two-ways ANOVA with Dunnett’s multiple comparisons test for the percentage of serum effect (*) and Turkey’s multiple comparisons test for the effect due to the percentage of serum within the same polyclonal serum. b Representative images, after performing the experiment three times with similar results, showing infection density (T. cruzi intracellular amastigotes, red) under live fluorescent microscopy after host cell DNA-Hoechst staining (blue), and before trypsinization. Images depict infections following incubation with various concentrations of polyclonal antisera IM1S and naïve serum. Scale bars = 100 µm. c Representative images display IgG binding in polyclonal sera, visualised through immunostaining using anti-mouse IgG-AF488 (green) in fixed and saponin-permeabilized trypomastigotes (red), with DNA stained using DAPI (blue). Nuclear (N) and kinetoplast (k) DNA are indicated by arrows. Naïve serum displayed no binding (negative control). Serum from a chronically infected (>100 days) mouse exhibited a dispersed binding pattern (positive control). Antiserum IM1S demonstrated high affinity for TcPOP in the secretory pathway, consistent the reported location. The zoomed image of a single trypomastigote shows a binding pattern within the parasite, corresponding to characteristic vesicles of the secretory pathway. The trypomastigote surface is highlighted by Wheat germ agglutinin staining (turquoise). Scale bars = 5 µm. (see Supplementary-movie 2.mov). d Schematic representation of the in vitro polyclonal antibody live-binding assay designed to capture the intermediate lysis effect induced by antiserum IM1S. Image produced with Biorender (https://BioRender.com/9tt05k9, academic licence) e Representative images from modified live-binding assays show experimental conditions where incubation temperature was reduced to 30 °C and serum concentration was 5% antiserum + 5% FBS At 5 min post-incubation, antibodies are detected binding to TcPOP in endocytic/exocytic vesicles near the flagellar pocket of the parasite, and trypomastigotes appear swollen. By 10 min, trypomastigotes exhibit internal leakage in multiple vesicles, followed by complete lysis by 15 min. Scale bars = 5 µm. All the microscopy experiments shown in this figure were performed independently three times obtaining similar results.