Fig 1.
(A) Scheme to illustrate the cohort of patients used for the gPhage selection, including an additional cohort of 21 patients used for ELISA validation studies. Patients were classified as responders or non-responders to benznidazole treatment based on their individual PCR results using two different criteria (see below for details). (B) Age distribution of patients before and after treatment.
Table 1.
Clinical characteristics for the cohort of patients.
Fig 2.
Identification using gPhage of Benznidazole associated-antigen.
Scheme illustrating the two-tier gPhage selection protocol.
Fig 3.
gPhage library construction and phage selection.
DNA inserts (fragments of T. cruzi genomic DNA) were cloned into the gPhage vector (top). Because inserts were randomly inserted into the vector into any of all possible reading frames, only a fraction of phage particles displays a T. cruzi-derived peptide (middle). However, with the successive rounds of biopanning, only phage displaying a T. cruzi-derived peptide are selected and enriched while the remaining phage particles are removed with wash (bottom).
Fig 4.
gPhage predicts treatment outcome.
(A) Bar graphs displaying the percentage of in-frame inserts for responders and non-responders according to both short- and long-term criteria. Responders showed a significant reduction in the percentage of in-frame inserts at T3 compared with T0 (Student T-test). (B) Pie graphs summarizing antigens recognized by responder and non-responder patients at T0 and T3 according to each criterion.
Table 2.
Antigens and epitopes preferentially recognized by patients’ IgG at the beginning of treatment (T0).
Table 3.
Antigens and epitopes preferentially recognized by patients’ IgG at the end of treatment (T3).
Fig 5.
(A and B) ELISA reactivity against select antigens for sera (T0 and T3) of patients classified as responders (blue) or non-responders (orange) according to each criterion. Sera from individual patients (dilution 1/200) (including 21 additional patients not used for the gPhage selection) were tested against synthetic peptides encoding each individual antigen CA-2 (B13), Microtubule-associated protein (MAP), Hypothetical protein TcG_12368 and R27-2 (peptide 4) (Wilcoxon paired-rank test: **** p<0.0001). (C) Reactivity of sera from individual peptides in comparison with OV assay. (D) Parasite load (quantified by PCR) for the cohort of 41 patients. OV: Orthos Vitros serology assay. R: responders. NR: non-responders. (Lsh = sera from patients with Leishmaniosis).