PLOS NEGLECTED TROPICAL DISEASES

RESEARCH ARTICLE

Anti-Trypanosoma cruzi antibody profiling in

patients with Chagas disease treated with
benznidazole assessed by genome phage
display
Luis Antonio Rodriguez Carnero1, Andréia Kuramoto2, Léa Campos de Oliveira3,
Jhonatas Sirino Monteiro1, João Carlos Setubal1, Edécio Cunha-Neto2,4, Ester Cerdeira
Sabino3☯‡*, Ricardo José Giordano ID1,4☯‡*
a1111111111
1 Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP, Brazil, 2 Heart
a1111111111
Institute (InCor) and Division of Clinical Immunology and Allergy, University of São Paulo School of Medicine,
a1111111111 São Paulo, SP, Brazil, 3 Department of Infeccious Diseases and Parasitology, University of São Paulo
a1111111111 School of Medicine and Institute of Tropical Medicine, University of São Paulo School of Medicine, São Paulo,
a1111111111 SP, Brazil, 4 Institute for Investigation in Immunology (iii), INCT, São Paulo, SP, Brazil

☯ These authors contributed equally to this work.

‡ These authors are joint senior authors on this work.
* [email protected] (RJG); [email protected] (ECS)

OPEN ACCESS

Citation: Rodriguez Carnero LA, Kuramoto A, Abstract

Campos de Oliveira L, Monteiro JS, Setubal JC,
Cunha-Neto E, et al. (2023) Anti-Trypanosoma Background
cruzi antibody profiling in patients with Chagas
disease treated with benznidazole assessed by There have been significant improvements in Chagas disease therapy and it is now widely
genome phage display. PLoS Negl Trop Dis 17(1): accepted that most patients with chronic disease might benefit from therapy. However,
e0011019. https://doi.org/10.1371/journal. there are challenges to monitor drug efficacy and cure for these patients, which are impor-
pntd.0011019
tant impediments for current and future therapies. Trypanosoma cruzi-PCR is highly vari-
Editor: Luisa Magalhães, Universidade Federal de able while IgG seroconversion takes decades yielding variable results depending on the
Minas Gerais, BRAZIL
antigen(s) used for the assay.
Received: August 9, 2022

Accepted: December 12, 2022 Methods and results

Published: January 6, 2023 We used the genomic phage display (gPhage) platform to perform a pairwise comparison of
Copyright: © 2023 Rodriguez Carnero et al. This is antigens and epitopes recognized by twenty individual patients with chronic Chagas disease
an open access article distributed under the terms before and after treatment with benznidazole. In total, we mapped 54,473 T. cruzi epitopes
of the Creative Commons Attribution License,
recognized by IgG from individual patients (N = 20) before benznidazole treatment. After
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
treatment, the number of epitopes recognized by all patients was significantly smaller
author and source are credited. (21,254), a reduction consistent with a decrease in anti-T. cruzi antibodies. Most of these
Data Availability Statement: All relevant data are
epitopes represent distinct fragments from the same protein and could, therefore, be
within the manuscript and its Supporting grouped into 80 clusters of antigens. After three years of treatment with benznidazole, we
Information files. observed a 64% reduction in the number of clusters of antigens recognized by patients (59
Funding: This work was supported by research clusters before versus 21 clusters after treatment). The most abundant antigenic clusters
grants from São Paulo Research Foundation recognized by patients correspond to the surface antigen CA-2 (B13) followed by the micro-
(FAPESP; www.fapesp.br) (grants: 2016/22.645-1
tubule associated antigen, which highlights the value of these epitopes in Chagas disease
and 2018/25.016-0 to R.J.G.), the National Council
for Scientific and Technological Development diagnosis. Most importantly, quantitative pairwise comparison of gPhage data allowed for
(CNPq; www.cnpq.br) (fellowship to L.A.R.C. the prediction of patient response to treatment based on PCR status.

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

141408/2019-7 and grant 307412/2017-2 to R.J. Principal finding

G.) and National Institutes of Health (NIH, grant
1U01AI168383-01 to E.C.S.). It was also financed Here, we compiled a list of antigens and epitopes preferentially recognized by Chagas dis-
in part by the Coordenação de Aperfeiçoamento de ease patients before and after benznidazole treatment. Next, we observed that gPhage
Pessoal de Nı́vel Superior - Brasil (CAPES; www. data correlated with patient PCR-status and could, therefore, predict patient response to
capes.gov.br) - Finance Code 001. The funders had
no role in study design, data collection and
treatment. Moreover, gPhage results suggest that overall, independent of PCR status, treat-
analysis, decision to publish, or preparation of the ment led to a reduction in the presence of T. cruzi-specific antibody levels and the number of
manuscript. antigens and epitopes recognized by these patients.
Competing interests: The authors have no
competing interests.
Conclusion
The gPhage platform use of unbiased library of antigens, which is different from conven-
tional serological assays that rely on predetermined antigens, is a contribution for the devel-
opment of novel diagnostic tools for Chagas disease.

Author summary
Chagas disease, caused by the single-celled parasite Trypanosoma cruzi, can be a life-treat-
ing and debilitating illness. Because there is no vaccine and currently the only two avail-
able drugs are most effective if used during the early acute stage of the disease, treatment
options for infected individuals are limited. Most individuals will only find out they have
Chagas disease during a routine medical examination or in blood bank while donating
blood, in which cases, they are already chronically infected. At this stage, treatment will
not undo clinical manifestations (i.e., cardiomyopathy) but may eliminate the parasite
and prevent disease progression. Currently, polymerase chain reaction (PCR) and serolog-
ical assays are the only diagnostic tools available, both with limitations in sensitivity and
accuracy. The lack of effective molecular markers thus prevents physicians to determine
whether a patient is parasite free and cured from the disease. It also has important impli-
cations for the development of new drugs to treat Chagas disease. Here, we studied the
reactivity of anti-T. cruzi antibodies in sera from a cohort of 20 patients that underwent
treatment for Chagas disease using a new method developed by our group named gPhage.
Using gPhage, we scanned all T. cruzi proteins to identify those that were reactive with the
antibodies from each individual patient before and after treatment. In sum, gPhage data
correlated with patient PCR-status and could, therefore, predict patient response to treat-
ment. It also revealed a new set of T. cruzi proteins that could be useful for the develop-
ment of future diagnostic methods.

Introduction
Caused by the protozoan parasite Trypanosoma cruzi, Chagas disease is a neglected tropical
disease that affects an estimated 6 to 8 million people worldwide, resulting in approximately
50,000 annual deaths [1]. In endemic areas, T. cruzi is mostly transmitted to human hosts by
infected feces of triatomine insect vectors [2]. In non-endemic regions, other forms of trans-
mission may still occur, such as congenital, transfusion, organ transplant or by ingesting con-
taminated food. Neither vaccine nor a fully-effective treatment is available for Chagas disease
[2,3].

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

The challenge to treat Chagas disease is in part due to the nature of the disease and its two
distinct phases: acute and chronic. Upon infection, the individual enters the acute phase of the
disease with high blood parasitemia but usually without specific symptoms. Therefore, it often
goes unnoticed and the great majority of patients is not diagnosed until later in life when they
are already with chronic Chagas disease [2,4]. The chronic phase is often asymptomatic and
the majority of infected people will eventually die of natural causes or other illnesses. Only a
fraction of them (approximately 30%) will develop, over the course of decades, the characteris-
tic heart or gastrointestinal Chagas disease (estimated at 9.2 events/1,000 infected person-
years) [5].
The two available drugs for Chagas disease therapy are benznidazole and nifurtimox.
Treatment with these drugs has shown a better performance when applied at the early stages
of the disease (acute phase) [2,6]. Unfortunately, this is seldom the case and most people will
only realize they have the disease much later in life, such as during a routine visit to their
doctor or by screening in blood banks. Currently, serological and polymerase chain reaction
(PCR) methods are used to determine presence and parasite load [7]. Nevertheless, due to
the very low parasitemia during the chronic stage of the disease, even the very sensitive PCR
method is not ideal, making it difficult, for instance, to assess drug efficacy. ELISA and
immunofluoresce assays are the most common methods to diagnose chronic Chagas disease,
with certain limitations for current available tests [8], but seronegativation may take decades
[2]. So, to date, there are no ideal molecular markers or methods to determine disease status
and cure.
Currently, benznidazole is the main treatment agent as it has shown to be less toxic [9] and
possibly more effective in reducing parasite load [6]. Nevertheless, it cannot revert loss of car-
diac function in patients that have already developed Chagas cardiomyopathy [9,10]. Hence,
the question remains whether or not parasite clearance detected by PCR is enough to establish
responsiveness to treatment. Considering that there is good evidence that the presence of T.
cruzi-specific antibody levels is inversely correlated with the active infection (determined by
PCR) in untreated patients [11,12], the humoral response to the parasite and the epitopes rec-
ognized by these antibodies may be important indicators of disease status.
To address these questions, we employed our recently developed genomic phage display
(gPhage) platform for antibody antigen identification and epitope mapping [13,14] to evaluate
the antibody response of Chagas patients that underwent benznidazole treatment. The gPhage
platform relies on an unbiased library of (all) T. cruzi antigens displayed on the surface of fila-
mentous phage, which are then presented to the IgG of patients with Chagas disease to identify
antigens and epitopes reactive with sera antibody. Using gPhage display, we have identified
immunodominant epitopes recognized by Chagas patients before and after antitrypanosomal
treatment, and observed that the reduction of reactivity to these markers could be associated
with benznidazole treatment efficacy.

Material and methods

Ethics statement
All participants in the study provided written informed consent before enrolment and the
study was approved by the Ethics Committee at School of Medicine, University of São Paulo
(approval 042/12).

Patient selection
For the gPhage screening, we selected 20 patients that underwent a full benznidazole treatment
course. Sera from each patient were obtained at the beginning of the treatment and 3 years

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

after baseline. PCR tests were performed before treatment, at baseline, and at 60 days, 6
months, 1 and 3 years after treatment. Data from PCR tests were used to classify patients as
responder or non-responder to treatment according to two distinct criteria (for details, see
Patient cohort in the Results and Discussion section). For validation studies by ELISA, sera
from an additional cohort of 21 patients were used in addition to sera from our initial cohort
of 20 patients (S1 Table).

Bacterial strains
Escherichia coli TG1 strain was used for library production/amplification and phage display
screening with patients.

gPhage library amplification and titration

DNA encoding the gPhage library [13] was transformed into electrocompetent E. coli bacteria
(TG1 strain, Lucigen) and growth in LB media until reaching Log phase (OD600nm 0.4–0.8).
Log phase bacteria were infected with M13KO7 helper phage (New England Biolabs) and cul-
tured overnight (ON) at 37˚C (250 rpm) in LB media containing kanamycin and ampicillin.
Next day, phage particles were purified using the PEG/NaCl method [13,15]. Aliquots of the
amplified phages were then kept frozen (-20˚C) until use. TG1 strain bacteria in Log phase was
infected with serial dilutions of the amplified gPhage library and cultured ON in LB agar plates
containing ampicillin, which was used to calculate the library titter by colony count and
expressed as Transducing Units (TU).

Adjustment of protein concentration in sera

Protein concentration in sera was quantified by Nanodrop (280 nm)(Thermo-Fisher Scien-
tific) and adjusted to a 60 μg/mL concentration with Phosphate Buffered Saline (PBS). Ali-
quots of diluted sera were frozen at -20˚C until use.

gPhage biopanning
Anti-human IgG specific for the Fc fragment (anti-Fc) (Jackson Immuno Research 1009-001-
008) was immobilized on microtiter plates wells (96 microwells) (ON at 4˚C), washed four
times with PBS and blocked with PBS containing 2.5% bovine serum albumin (PBS/BSA) for
2h at room temperature (RT). Well coated with anti-Fc IgG were then used to capture IgG
from individual patient’s sera. To perform the two-tier biopanning, 50 μL of the pre-clearing
sera (T0 or T3) were added to the anti-Fc coated-wells, incubated for 2h at room temperature
and the wells washed 4 times with PBS. The gPhage library (or pool of phage from the previous
round of selection) was then added to each well containg the captured IgG at a concentration
of 1010 TU in 50 μL and incubated for 2h at RT. After the incubation period, unbound phage
was recovered and transferred to a new well coated with anti-Fc and pre-incubated with the
sera from the same patient at the second time-point (T3 or T0). After 2 hours of incubation at
RT, wells were washed 10 times with 200 μL of PBS 0.5% Tween-20 (PBST) and phage bound
to the target IgG was recovered by bacterial infection at 37˚C for 30 min (50 μL of E. coli TG1
strain in log phase). Infected bacteria were diluted in 2xTY media containing ampicillin
(50 μg/mL) and co-infected with helper phage M13KO7 (1010 TU). Kanamycin (50 μg/mL)
was then added to the cell cultures and bacteria and phage cultured ON at 37˚C (250 rpm, rev-
olutions per minute). On the following day, bacteria were centrifugated at 8,000g for 10 min
and phage particles recovered from the supernatant by the PEG/NaCl method [15]. Phages
were tittered and 5x109 TU of phage per well were used for next rounds of selection. In total,

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

four rounds of selection were performed. After selection, the pool of phage particles was used
for large-scale sequencing. Between the second and fourth rounds of selection, aliquots of the
bacteria infected with bound phages were separated for quantification of phage recovery by
colony count (quantification of bound phages enrichment).

Large-scale DNA sequencing

Large-scale DNA sequencing was performed using MiSeq Reagent Kit v2 (500 cycles) on an
Illumina MiSeq equipment at the Center for Advanced Technologies in Genomics at Institute
of Chemistry, University of São Paulo. Sequencing was performed as previously described
[13,16]. In brief, specific oligonucleotide primers were used to amplify by PCR the gene VIII
region encoding the T. cruzi antigens from phage isolated from each patient. The phage popu-
lation to be sequenced was chosen according to the enrichment data in third or fourth rounds
of selection (highest enrichment). The indexation was performed in two steps: one initial PCR
to add the barcodes and a second using the PCR Nextera kit to complete the process. We used
four different forward and reverse primers containing zero to three degenerated bases to add
the diversity necessary for amplicon sequencing with the Illumina platform. Phages from third
or fourth rounds were adjusted to 108 TU/μL and used as template in 25 μL PCR reactions
with Kappa HiFi Polymerase (Kapa Biosystems, Roche) for 20 cycles (melting: 98˚C for 20 s;
annealing: 65˚C for 15 s; extension: 72˚C x 20s). PCR products were then purified with QIA-
GEN PCR purification kit and quantified in Nanodrop (260 nm). An 8 cycles PCR was per-
formed to add the index adaptors (barcodes) with the Nextera XT kit (Illumina) (melting:
98˚C for 20s; annealing: 55˚C for 15s; extension 72˚C for 30s). The final PCR products were
purified with QIAGEN PCR purification kit and quantified by PicoGreen (Thermo-Fisher Sci-
entific). The size of the indexed PCR products was evaluated by estimation of PCR migration
in ImageJ and adjusted to 4 nM concentration. Equal volumes of the adjusted concentration
products were mixed and the 4 nM resulting library quantified by qPCR using a KAPA Library
quantification kit (Kapa Biosystems, Roche). The library was denatured (0.2M NaOH, 95˚C
for 5 min) and sequenced with a MiSeq Reagent Kit v2 (500 cycles) on an Illumina MiSeq
equipment.

Bioinformatic analysis
Bioinformatic analysis was performed as reported with a few modifications [13]. Pair assem-
bling was performed with PEAR [17] and DNA sequences extracted and sorted using the bar-
code sequence. Singletons were discarded and remaining sequences aligned to four T. cruzi
genomes [CL Brener (GenBank: GCA_000209065.1), DM28c (GenBank: GCA_003177105.1),
marinkellei (GenBank: GCA_000300495.1), Sylvio X10 (GenBank: GCA_000188675.2)] using
BLAST-like alignment tool (BLAT) [18]. Inserts with less than 95% identity were discarded as
well as those containing stop codons or not in the correct frame to produce a pVIII-fused pep-
tide. Remaining inserts were translated and the resulting peptides aligned to the proteome
(derived from the same T. cruzi genomes above) with BlastP [19]. Peptides with at least 60%
identity with T. cruzi proteins were considered for next steps. In order to cluster the epitopes,
identified peptides were sorted by abundance (decreasing order) and the first peptide (seed)
removed from the list and compared with the remaining peptides using the FuzzyWuzzy pack-
age [13]. Peptides with at least 80% sequences similarity with the seed were removed from the
list and included in the growing cluster. The pipeline was performed for each patient individu-
ally until all peptides were clustered. The cluster consensus sequences were submitted to BlastP
to identify the related T. cruzi antigen.

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

Enzyme-Linked Immunosorbent Assay (ELISA)

Peptides (B13:FGQAAAGDKPPLFGQAAAGDKPSL; MAP:YKRALP-
QEEEEDVGPRHVDPDHFRSTT; Hypothetical protein TcG_12368:GGFGSATTTST-
PAAGGFGS-AAHTSTPAVG; R27-2: KVAEAEKQRAAEATKVAEAEKQRAA; Putative
Trans-sialidase RNE97461.1: YIDGKSLGEEEVPLTGEKPLELF) were synthetized (Chinese
Peptide Company) and used for the ELISA tests. Peptides were diluted to a 50 μg/mL concen-
tration in 50 mM carbonate buffer, pH 9.0. Then, 50 μL of diluted peptides were immobilized
ON in 96-well microtiter plates. Next day, the wells were washed 3 times with PBST, blocked
with PBS/BSA for 2h at RT. Patient sera at 1/200 dilutions were added to the wells and incu-
bated with the peptides for 1h at 37˚C. Wells were washed 5 times with PBST, 50 μL of second-
ary anti-Human IgG antibody conjugated to horseradish peroxidase (Sigma, A6029) 1/5000
dilution added and the wells incubated at 37˚C for 1h. ELISA were quantified using the OPD
substrate (SigmaFast OPD, P9187). The reaction was stopped using 50 μL of H2SO4 per well
and the absorbance read at 420 nm. We used sera from 12 healthy donors and 6 Leishmaniasis
patients as negative controls for ELISA. Cut-off values of O.D.420nm were determined by com-
parison with control samples (B13: 0.11; MAP: 0.06; Hyp TcG_12368: 0.06; R27-2: 0.07; TS
RNE 97461.1: 0.07).

Statistical analysis
Software GraphPad Prism7 was used for graphical and statistical analyses. Data with normal
distribution (D’Agostino-Pearson or Kolmogorov-Smirnov test) were analyzed by paired Stu-
dent’s T-test. Data that did not show a normal distribution were analyzed by Wilcoxon signed-
rank test.

Results and discussion

Patient cohort
To identify and compare T. cruzi antigens recognized by patients with Chagas disease before
and after treatment, we selected a cohort of 20 patients with variable ethnic origins (but
mostly male) with median age of 47 (min. 35, max. 66 years) (Fig 1A and 1B), who com-
pleted at least 75% of the benznidazole treatment (Tables 1 and S1) (19/20 patients com-
pleted the full treatment). All patients had chronic disease with varied clinical status at the
beginning of the treatment. Before treatment, patients were tested for the presence of the
parasite by multiple PCR assays and all patients selected for this study presented at least one
positive result at the pre-treatment screening and/or the PCR test performed at baseline.
Treatment lasted for 60 days and PCR screenings were performed at baseline (Time 0, T0)
throughout the first year and then 3 years after the beginning of treatment (year 3, T3) with
variable results for each patient (Table 1). We used data from the multiple PCR tests that
were performed during the follow-up period to determine patient’s response to benznida-
zole: responder (PCR negative) or non-responder (PCR positive). Because there is not yet a
definite guideline of how to assess whether a patient is considered free of disease, we opted to
employ two distinct criteria. For the first criterion, if a patient was PCR negative at T3, he/
she was considered a responder. According to criterion #1, 16 patients were responders
while 4 remained as non-responders (Table 1). For the second criterion, if any of the PCR
tests were positive during the follow-up period, the patient was considered a non-responder.
Applying criterion #2, we observed that 9 patients were responders while 11 remained as
non-responders (Table 1). Age distribution did not correlate with patients’ response to treat-
ment (Fig 1B).

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

Cohort 1 Cohort 2 [Cohort 1 + 21 patients]

(panning selection). (ELISA)
n = 20 n = 41
A
PCR-positive
Chagas Disease patients
(pre-screening and Time 0)
Criterion 1
(long-term)

n = 16 n=4 n = 32 n=9
60-day PCR-negative PCR-positive PCR-positive
benznidazole three years PCR-negative
three years three years three years
treatment after the beginning after the beginning after the beginning after the beginning
of the treatment (T3) of the treatment (T3) of the treatment (T3) of the treatment (T3)
3 years
later Responders Non-responders Responders Non-responders

Day 0 T3

Day 60 Criterion 2
(short-term)

n = 20 n = 11 n=9 n = 10 n = 31
At least one Only negative PCR At least one
Only negative PCR results between positive PCR
results between positive PCR
result between day 60 and T3 result between
day 60 and T3 day 60 and T3
day 60 and T3

Criterion 1 (long-term) - Age distribution Criterion 2 (short-term) - Age distribution

80 80
p > 0.05 p > 0.05
B 80
66 years
60 60

Age (years)
Age (years)

60
Age (years)

40 40 40
35 years

20 20 20

0
Patient 0 0
Responders Non-responders Responders Non-responders
G G
Fig 1. Patient cohort profile. (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.
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gPhage display screening

We used the gPhage technology [13,14] to map the antigen profile of each individual patient
before and after treatment. gPhage is a combinatorial technology that allows for the identifica-
tion of antigens and epitopes recognized by antibodies. This is achieved by fragmenting the
whole genome of the parasite and inserting each individual fragment into the filamentous
phage genome. The end result is a large collection of phage particles, each displaying a unique
peptide encoded by the parasite genome. The gPhage library used in this study had approxi-
mate 100-fold coverage of the corresponding T. cruzi proteome [13]. If an immobilized anti-
body from a Chagas patient recognizes the antigen/epitope displayed by an individual phage
particle, it is then captured and can be recovered and the antigen identified by DNA sequenc-
ing (Fig 2).
To map the antibody antigen profile of Chagas patients before and after benznidazole treat-
ment, sera from each patient was collected at the beginning of the treatment (T0) or 3 years
after baseline (T3). In order to enrich for the identification of antigens specifically recognized
by the target antibody (i.e., T0 or T3), a gPhage library pre-clearing step was included; thus, we
performed for each individual patient a two-tier panning procedure. For instance, we first pre-

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

Table 1. Clinical characteristics for the cohort of patients.

Patient Gender Clinical status ECG ECG PCR result Treatment outcome
(base) (3rd year) 60 days 6 months 12 months 3 years Criterion #1 Criterion #2
1 M C Normal Ventricular - - - + NR NR
repolarization
2 M C Altered Right Bundle - - + + NR NR
Branch Block
3 M I Altered Left Bundle - + - + NR NR
Branch Block
4 F C Altered Right Bundle - + - + NR NR
Branch Block
5 M C Normal Ventricular - - - - R R
repolarization
6 M C Altered - - - - - R R
7 F I Normal Normal - - - - R R
8 F I Altered - - - - - R R
9 M Mi Altered Normal - - - - R R
10 M I Normal Normal - - - - R R
11 M I Normal Atrioventricular - - - - R R
block
12 M C Altered Right Bundle - - - - R R
Branch Block
13 M Mi Normal Normal - - - - R R
14 M C Normal Ventricular - - + - R NR
repolarization
15 F C Normal Normal + - - - R NR
16 M I Normal - + + - - R NR
17 F I Altered Ventricular + + - - R NR
repolarization
18 M C Altered Right Bundle + - - - R NR
Branch Block
19 M C Altered Ventricular - + - - R NR
repolarization
20 M I Normal Sinus rhythm + + - - R NR

Gender: male (M), female (F). Clinical Status: cardiac (C), indeterminate (I), mixed (Mi) form of the disease. Treatment outcome after 3 years: non-responder (NR) or
responder (R). PCR for T. cruzi presence were performed at 60 days, 6 months, 12 months and 3 years after the beginning of treatment. ECG: Electrocardiogram.

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cleared the gPhage library using serum at baseline (T0) before performing the phage selection
on IgG from serum at year 3 (T3), and vice-versa (Fig 2). In all cases, we performed four
rounds of selection. We then selected either the 3rd or 4th round (the round with the highest
enrichment in phage particles captured by the target IgG in comparison with the 2nd round)
for large-scale DNA sequencing in order to identify the antigens and epitopes selected during
the biopanning.

Large-scale DNA sequencing and Bioinformatic analysis

Following the gPhage selection, we used large-scale sequencing [16] and our in-house bioin-
formatic pipeline to identify antigens and epitopes recognized by IgG from each individual
patient, at T0 and T3 [13]. In summary, we observed that on average there was significant
phage enrichment for most patients when comparing the round used for sequencing (3rd or
4th) with the second round (median 7, min = 0.13 and max = 32) (first round was not

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

Cleared library
T0 IgG Escherichi coli T. cruzi
T3 IgG epitope

Captured
gPhage Library IgG

Anti-Fc

Cleared Washing of
libraries unbound
incubation phages
with target IgG T. cruzi
epitope

Captured
IgG

Anti-Fc
Phage Binding to target IgG Bacteria Infection
Step I. Naive Library Pre-clearing Step II. Panning (first round)
(before first round of selection)

New phage
population

Step III.Selection Cycles Step IV. Sequencing

Phage amplification
by growing infected
bacteria

Step V. Bioinformatic
analysis
Fig 2. Identification using gPhage of Benznidazole associated-antigen. Scheme illustrating the two-tier gPhage selection protocol.
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quantified to minimize the loss of putative antigens). We obtained on average 228 thousand
reads per sample (min. = 107,642, max. = 386,946 reads) (S2 and S3 Tables). As we did in our
previous study [13,14], all individual reads had to be present at least twice within a given sam-
ple. By removing all singletons, we minimized sequencing errors and increased our confidence

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

in the final list of antigens and epitopes used for this study. Insert sequences were also aligned
to the T. cruzi genome and those that did not have at least 95% identity were discarded.
Because of the characteristics of the gPhage library (phagemid), not all phage particles dis-
play an exogenous peptide. This is because during library construction, inserts are randomly
inserted into the phage genome, and not all are in the correct reading-frame to produce a real
T. cruzi epitope fused to the pVIII chimeric protein (Fig 3). In order to be effectively displayed
in the bacteriophage surface, the insert has to be in frame with the vector’s signal peptide
sequence (5’) and the bacteriophage gene VIII (3’) (Fig 3). But it is also possible for a T. cruzi
DNA insert cloned in the wrong reading-frame to have an open-reading frame (ORF) that
results in the display of an artefactual non-T. cruzi peptide (Fig 3). These inserts were mostly
removed by aligning the encoded peptide against the T. cruzi proteome.
Because our initial gPhage library was constructed by randomly cloning fragments of the
parasite’s genome, it contained a modest 8.6% of phage particles displaying a T. cruzi antigen
(nevertheless, the coverage of our gPhage library is over 100-times the parasite’s encoded-pro-
teome). Hence, upon selection, we observed an enrichment in the number of phage particle
displaying a T. cruzi antigen: on average, 51% for both patients in T0 and T3 (min = 0,
max = 100%) (Fig 3 and S2 and S3 Tables). This indicates that the gPhage selection was suc-
cessful: phage particles that did not carry a T. cruzi antigen were washed away during the selec-
tion process, while those that contained and epitope recognized by the patient’s IgG were
captured and enriched during the successive rounds of selection (Figs 2 and 3). However,
because of our two-tier selection process, enrichments were lower than we have observed pre-
viously [13]. On the other hand, this decrease was expected, considering that we performed a
pre-clearing step using sera from a Chagas patient that clearly reduced the number of antigens
recovered in the second step of the selection.
After our bioinformatic pipeline processing was complete, we used the enrichment data as
proxies for patient sera immunogenicity. For both criteria, we observed a positive correlation
between the percentage of in-frame inserts and the PCR status of the patients. Patients that
were classified as responders based on their PCR results according to both criteria showed a
significant (p = 0.0081, criterion #1 or p = 0.0246, criterion #2) lower percentage of in-frame
inserts recovered by T3 IgG (mean = 16.2, criterion #1 and mean = 23.1, criterion #2) in com-
parison with T0 (mean = 45.9 for criterion #1 or mean = 57.5 for criterion #2) (Fig 4A), which
may be related to a sera decrease of T. cruzi-specific antibodies. No significant difference
(p > 0.05) was observed for non-responder patients when comparing T3 (mean = 42.9, crite-
rion #1 and mean = 23.1, criterion #2) with T0 (mean = 33.7, criterion #1 and mean = 32.0, cri-
terion #2) (Fig 4A). These data indicate that gPhage may be useful to predict the outcome of
treatment and that antigens captured during our biopanning procedure may reflect patient’s
disease status.

Antigen profile before and after benznidazole treatment

Given the observed correlation between our gPhage selection and the PCR status of patients,
we next compared the antigen profile of responders and non-responders, before and after
treatment. Because there was no significant enrichment of in-frame gPhage particles display-
ing a T. cruzi antigen in comparison with the naïve gPhage library for some of the patients, we
only considered data from patients with a positive selection of at least twice (17.2%) the value
of in-frame inserts present in the initial naïve gPhage library (8.6%). So, for these analyses we
had data from 13 patients at T0 and from 7 patients at T3.
Applying our bioinformatic pipeline [13], we aligned the translated inserts to the T. cruzi
proteome and considered only sequences with at least 60% of identity (to allow for antigen

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

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).
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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

variability due to different strains and the numerous multigene families). This resulted in
75,727 T. cruzi-derived peptides that were recognized by all patients (a total of 54,473 peptides
at T0 and 21,254 peptides at T3). However, many peptides represent distinct fragments from
the same antigens. Thus, we next performed a clusterization step to generate a list of non-
redundant antigens and epitopes, as previously described [13,14]. This resulted in 80 clusters
of antigens (59 at T0 and 21 at T3) (S1 Data). Here, it is interesting to note the reduction
(from 59 to 21, or 64%) in the number of antigens/epitopes recognized by patients after 3 years
of treatment with benznidazole. These data agree with the reduction in the percentage of in-
frame inserts (Fig 4A and S2 and S3 Tables) and previous reports of seronegativation follow-
ing benznidazole treatment [20–22].
We observed that for responders and non-responders, the most predominant antigenic
cluster recognized by patients was the CA-2 surface protein (B13 epitope), with 24,537 pep-
tides forming 17 clusters at T0 (4 for non-responders and 13 for responders) and 19,407 pep-
tides constituting 12 clusters at T3 (3 for non-responders and 9 for responders) (Tables 2 and
3 and Fig 4B). Another well-represented antigen was the microtubule-associated protein
(MAP), mainly recognized by patients within the responder group at T0, with 8,698 peptides
forming 12 clusters for responder patients at T0. Only two patients (#10 and #11) showed
non-CA2 antigens as immunodominant and predominant clusters: Mucin (TcMucII) and
MAP, respectively. Although highly immunogenic and the largest multigene family, members
of the trans-sialidase were not among the most abundant antigens/epitopes identified, with the
notable exception of one patient (#12 at T3) who had a trans-sialidase antigen as the immuno-
dominant epitope. Interestingly, this particular trans-sialidase epitope is a long-tandem repeat
(with over 50 repetitions of the identified epitope—protein ID# PBJ71599.1), a common ele-
ment in our gPhage screening. Finally, the R27-2 antigen, another long-tandem repeat antigen
that shares similarity with the CRA1/2 antigens [23], and several hypothetical proteins were
also recognized by IgG of multiple patients, mostly at T3. In sum, our data are in agreement
with previous observations that benznidazole treatment has a significant effect on the patient’s
antibody response, reducing anti-T cruzi antibodies although recognized antigens appear to
vary between individuals, with no recognizable signature antigens for responders and non-
responders.

Epitope validation
Based on the antigen profile we obtained, we next tested by ELISA (Fig 5) the reactivity of sera
from patients who were classified as responders and non-responders. For that end, sera col-
lected at baseline (T0) and T3, including an additional cohort of 21 new patients treated with
benznidazole but for which we have no gPhage data (S1 Table), were tested against the two
immunodominant antigens identified in our study, CA-2 (B13) and MAP. We also include
two minor epitopes, the R27-2 and the hypothetical protein TcG_12368. Synthetic peptides
encoding each epitope were immobilized on microtiter wells and reactivity of sera from each
patient (T0 and T3) was quantified by ELISA. We observed that, overall, sera from all patients
reacted with at least one of the antigens and that reactivity was consistently lower with sera col-
lected at T3 than with sera collected at the beginning of treatment (T0) (Figs 5A, 5B and S1).
These results agree with previous studies that indicate a decrease in anti-T. cruzi antibodies in
plasma upon benznidazole treatment [21,22] and with our own gPhage results. All antigens
showed null or very low reactivity towards the control samples (blood bank donors that tested
negative for Chagas disease) or patients with Leishmaniasis, indicating that the markers identi-
fied by gPhage were specific for Chagas disease (Fig 5A). A similar profile was observed for
B13, MAP, R27-2 and Hypothetical protein TcG_12368. Next, we compared our ELISA results

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

In-Frame inserts for Non-responders In-Frame inserts for Responders

A (criterion 1: long-term) (criterion 1: long-term)
100 100
p < 0.05

% In-Frame Inserts
80

% In-Frame Inserts
80
*
60 60

40 40

20 20

0 0
T0 (baseline) T3 (third year) T0 (baseline) T3 (third year)
Time Time

In-Frame inserts for Non-responders In-Frame inserts for Responders

(criterion 2: short-term) (criterion 2: short-term)
100 100
p < 0.05
% In-Frame Inserts 80

% In-Frame Inserts
80
*
60 60

40 40

20 20

0 0
T0 (baseline) T3 (third year) T0 (baseline) T3 (third year)
Time Time

B. All patients Responders Non-responders

2.3% 3.7% 4.1 %
1%

Criterion 1 24.3%
(long-term)
2.5%
2.1% 1.6% 68.7 % 95.4 %

16%

T0 5.9% 0.8%
2.4 %
1.6%3.6%
77.8 %

Total = 54473 Criterion 2

(short-term) 51.3 %
37.5 %
96.4 %

Putative surface antigen 2 (CA-2)

Microtubule-associated protein
Trans-sialidase
TcMucII
Hypothetical protein
Other 12.6%

Criterion 1
(long-term)
86.5 % 99.4 %
7.9%

T3
91.3 % 15.3%

Criterion 2
(short-term)
Total = 21254
84.1 % 99.1 %

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

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.
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with data obtained using the Orthos Vitros (OV) commercial kit, which showed a similar
decrease in antibody reactivity (Fig 5B and 5C), with a reduction of reactivity not necessarily
correlated with the parasite relapse in non-responder patients (Fig 5D). However, although
there was a similar reduction in the reactivity towards T. cruzi antigens in sera collected at T3
and T0 among responder and non-responders (quantified by the ratio T0/T3), data from
ELISA and OV could not predict patient outcome with regard to benznidazole treatment
regardless of the criteria used to assess patient response to treatment (Figs 5B, 5C and S1).
The decrease in reactivity at T3 in comparison with T0 was 73.5% for B13, 61.7% for MAP,
66.1% for Hyp TcG_12368, and 70.1% for R27-2. It is interesting that both serological assays
were not able to predict patient outcome (clearance in parasitemia) as gPhage did, although
both methods correctly predicted a reduction of parasite load induced by benznidazole treat-
ment based on a reduction in anti-T. cruzi antibodies (Fig 5D).

Conclusions and limitations of the study

In this work, we studied the landscape of the antibody response of individual patients with
Chagas disease following a full course treatment with benznidazole. We show that gPhage
technology [13] can be utilized to screen and quantify the antibody response of Chagas disease
patients and, in agreement with previous reports [20–22], we observed that benznidazole treat-
ment led to a significant reduction of anti-T. cruzi antibodies and the overall number of epi-
topes/antigens recognized by sera IgG. Our study also highlights the diagnostic value of
previously identified antigens/epitopes (CA-2 and MAP) as possible markers for cure. Interest-
ingly, however, the in-frame data from gPhage could be used as proxies to predict treatment
outcome, a result that neither ELISA nor the OV data could achieve using samples from the
same cohort of patients. One possible explanation for this is that gPhage relies on an unbiased
library containing all (or most of the) antigens encoded in the parasite’s genome to probe each
patient individually. In that sense, gPhage is similar to a precision medicine diagnostic tool, as
opposed to serological assays that rely on a single or a combination of pre-determined anti-
gens, some of which not all patients might react to.
Limitations of the present study are the relatively small cohort of patients and the lack of
specific guidelines for determining whether a patient is a responder or a non-responder to
benznidazole treatment. Hence, we proposed and evaluated two criteria to assess patient
response to treatment, which we defined as long- and short-term criteria. Most importantly,
regardless of the criterion used, the gPhage data could be used to predict treatment outcome.
Therefore, the use of two criteria mitigated the limitation of our small cohort.
Another aspect that merits specific comments is the strategy to perform the gPhage selec-
tion using a pre-clearing step. By removing from the gPhage library antigens that reacted with
anti-T. cruzi antibodies found in a given serum sample (i.e., before treatment) we favored the
selection of antigens specific for antibodies found in the next paired serum sample (i.e, after
treatment). However, this strategy seems to have reduced significantly the number of antigens
mapped, including samples for which we recoved very few phage particles and, therefore,
could not identify any preferentially selected antigen or epitope. On the other hand, this strat-
egy may have allowed for a more direct comparison (quantification) of gPhage data (before/
after treatment) for each individual patient. But because it limited the number of antigens

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

Table 2. Antigens and epitopes preferentially recognized by patients’ IgG at the beginning of treatment (T0).
Patient Antigen Accession Frequency (%) Consensus sequence
1 <17.2% in-frame inserts
2 Putative surface antigen 2 (CA-2) PWU84979.1 95.69 GQAAAGDKPSPFGQAAAGDKPPPFGQAAAGDKPSPFGQAAAGDK
Trans-sialidase, putative, partial EKF99511.1 3.73 KASVHVDGESLGNEEVPLTGEKPPE
Trans-sialidase, putative, partial EKF98285.1 0.40 TSLGEEEVPLTGEAPLGLV
Putative trans-sialidase PWU90932.1 0.13 PDSFSSTNVSGGVDAAPAPSSTASG
Trans-sialidase, putative EKG00226.1 0.05 APSSTASGETKIPSELNATVPSDHDILLEFRELA
3 Putative surface antigen 2 (CA-2) PWU84979.1 99.69 QAAAGDKPPPFGQAAAGDKPSPFEQAAAG
Putative surface antigen 2 (CA-2) PWU84979.1 0.31 GDRPSPFEQAAAGAKPSPFGQ
4 Putative surface antigen 2 (CA-2) PWU84979.1 40.49 AAGDKPPPFGQAAAGDKPSPFGQAAAG
Mucin TcMUCII XP_808990.1 23.93 PSTTTTEAPTTTTTRAPSRLREID
Mucin TcMUCII, putative EKF29049.1 9.82 NTARNTEAPITTTTTRAPSRFREID
Hypothetical protein TCSYLVIO_010170 EKF98926.1 9.82 HDIYHQRNPNSSGSVGRRGGVWGRTVSPASTEQGL
Monoglyceride lipase, putative, partial EKF27476.1 4.91 VPLTARYGAEMMRAIDD
Hypothetical protein TCSYLVIO_009815 EKF99266.1 3.68 ETVDALNEKVWTAEFRQIDTE
Hypothetical protein MOQ_008444, partial EKF27823.1 2.45 TDTTPLVIVGPETSVAPVAAQRAIDTV
Putative 60S ribosomal protein PWU84687.1 1.84 VTGTAMPRGMQAFPLRDSAETI
Mucin TcMUCII, putative EKF29049.1 1.84 AGNTEAPATATTRAPSRLREID
Hypothetical protein XP_822001.1 1.23 RQRLVDPSEPPTSPASTEMDETGKAST
5 Putative surface antigen 2 (CA-2) PWU84979.1 70.41 GQAAAGDKPSPFGQAAAGDKPSPFGQAAAGDKPSPFGQAAAGDK
Putative microtubule-associated protein PWU97874.1 24.05 VDPSAYKRALPQEEEEDVGPRHVDPDHFRSTTQ
Hypothetical protein C4B63_133g26 PWU86111.1 4.87 PFKSVFGAPSSTDAKPPAESPFKS
Microtubule-associated protein, partial XP_803031.1 0.32 MGPSAQNYDTQEEEDVGPRHVDPDHFRST
Hypothetical protein C4B63_76g64 PWU88332.1 0.20 ATHERAVEALAAEEDAARGQLVGGE
Hypothetical protein C4B63_76g64 PWU88332.1 0.06 AAVDELGEAFRSATHERAVEALAAEEDA
Surface antigen 2 (CA-2) XP_818927.1 0.04 SPFGQAAAGDKPPLFGQGTVFDAS
Hypothetical protein XP_813515.1 0.02 DAKPPAESPFKGGFGAPSSTVAKPPGESPFKN
Putative surface antigen 2 (CA-2) PWU84979.1 0.02 AAGDKPPPVEQAAGGDRPSPFEQA
Putative microtubule-associated protein PWU97874.1 0.02 YKRALPEEGQGDLGPRQVDPDHFRSTTQDA
6 putative surface antigen 2 (CA-2) PWU84979.1 76.33 SPFGQAAAGDKPPPFGQAAAGDKPSPFGQAAAGDKPPL
hypothetical protein C4B63_9g493 PWU99324.1 21.11 SAPAAGGFGSATTTSAPAVGGFGSATT
putative mucin TcMUCII PWU85205.1 2.17 TPSTTTTGTPTTTTTRAPSRLREIDS
putative microtubule-associated protein PWU97874.1 0.28 LPQEEQEDVGPRHVDPDHFRSTTQDAY
hypothetical protein C4B63_9g493 PWU99324.1 0.06 VGGFGSAAHTSTPGVGGFGSATTT
mucin TcMUCII, putative EKF29049.1 0.04 PINTAGKTEAPTTTTITHAPSRLREID
7 Too few peptide sequences
8 Putative surface antigen 2 (CA-2) PWU84979.1 100.00 DKPPPFGQAAAGDKPSPFGQAAAGDKPSPF
9 <17.2% in-frame inserts
10 Mucin TcMUCII XP_821913.1 95.90 TEASTTTTTRAPSRLREID
Mucin TcMUCII XP_820005.1 2.97 TTTTTTEAPNTTIPRAPSRLREID
Mucin TcMUCII, putative EKG05373.1 0.85 TTTTTTADPTTTSARTPSRLREID
Mucin TcMUCII, putative, partial EKG03739.1 0.28 TTTTTTSAPEAPSNTTMNTEAPTPTTSRAPLRLREIDV
11 Putative microtubule-associated protein PWU97874.1 93.43 ALPQEEEEDVGPRHVDPDHFRSTTQ
Putative surface antigen 2 (CA-2) PWU84979.1 6.45 QAAAGDKPPPFGQAAAGDKPSPFGQAAAGDKPS
Flagellar attachment zone protein 1 PWU95940.1 0.10 EELEQKAAENERLAEELEQKAAENE
Putative microtubule-associated protein PWU97874.1 0.01 EEQEDVGPRHVGPDQFPPTTQDAYRPVDPS
12 Putative surface antigen 2 (CA-2) PWU84979.1 71.29 AAGDKPSPFGQAAAGDKPSPFGQAAAGDKPLPFEQA
Putative trans-sialidase PWU87189.1 28.31 MPAGTSEEGSRDDSPMPAGASEEGSRDD
Putative microtubule-associated protein PWU97874.1 0.24 SAYKRALPQEEEEDVGPRHVDPDHFRST
Putative surface antigen 2 (CA-2) PWU84979.1 0.16 AAGEKLPFGKAAAGDKPPPFGQA
13 Putative surface antigen 2 (CA-2) PWU84979.1 96.10 SPFGQAAAGDKPSPFGQAAAGDKPSPFGQA
Putative microtubule-associated protein PWU97874.1 1.74 LPQEEQEDVGPRHVDPDHFRSTTQ
Hypothetical protein XP_820768.1 1.67 DSVTLTSLWSSRTAQARPASMRLVDGV
Hypothetical protein XP_810212.1 0.49 PAVGGFGSATTTSAPAAGGFGSATTTSAPA
14 Putative surface antigen 2 (CA-2) PWU84979.1 96.84 GQAAAGDKPPPFGQAAAGDKPSPFGQ
Putative surface antigen 2 (CA-2) PWU84979.1 3.16 DKPSPFGQVAAGEKPPPFGQAAAGEK
15 <17.2% in-frame inserts
(Continued )

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

Table 2. (Continued)

Patient Antigen Accession Frequency (%) Consensus sequence

16 Putative surface antigen 2 (CA-2) PWU84979.1 99.95 GQAAAGDKPPPFGQAAAGDKPSPFGQA
Retrotransposon hot spot (RHS) protein EKF99800.1 0.05 MNCTPCGPFCWGMAGGYVGWNYCLRQHGRRLTFM
17 Putative surface antigen 2 (CA-2) PWU84979.1 97.31 AAGDKPPPFGQAAAGDKPPPFGQAAAGDKP
Putative microtubule-associated protein PWU97874.1 2.66 ALPQEEEEDVGPRHVDPDHFRSTTQD
Putative microtubule-associated protein PWU97874.1 0.01 SAYKRALPQEEEGGVGPGPVDPAHFRSP
Putative microtubule-associated protein PWU97874.1 0.01 SAYKRALPQEEEEDVGPGQVDPDQFRWT
Putative microtubule-associated protein PWU97874.1 0.01 SAYKRALPQEKEGDGGPGHVVPDHFRST
18 <17.2% in-frame inserts
19 <17.2% in-frame inserts
20 <17.2% in-frame inserts
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Table 3. Antigens and epitopes preferentially recognized by patients’ IgG at the end of treatment (T3).
Patient Antigen Accession Frequency (%) Consensus sequence
1 <17.2% in-frame inserts
2 Putative surface antigen 2 (CA-2) PWU84979.1 100.00 FGQAAAGDKPPPFGQAAAGDKPSPFGQ
3 Putative surface antigen 2 (CA-2) PWU84979.1 60.12 AAGDKPPPFGQAAAGEKPSP
Putative microtubule-associated protein PWU97874.1 27.98 VDPDHFRSTTQDAYRPVDPSAYK
Putative surface antigen 2 (CA-2) PWU84979.1 11.31 FGQAAAGDKPPPFGQA
Putative trans-sialidase PWU87189.1 0.60 MPAGTSEEGSRDDSSMPAGT
4 <17.2% in-frame inserts
5 <17.2% in-frame inserts
6 <17.2% in-frame inserts
7 <17.2% in-frame inserts
8 <17.2% in-frame inserts
9 <17.2% in-frame inserts
10 <17.2% in-frame inserts
11 Putative surface antigen 2 (CA-2) PWU84979.1 94.25 SPFGQAAAGDKPPPFGQAAAGDKPSPFG
Putative microtubule-associated protein PWU97874.1 4.60 ALPQEEEEDVGPRHVDPDHFRSTTQDA
Putative surface antigen 2 (CA-2) PWU84979.1 0.57 DKPSLFGQAAAGDNPSPFGQAAAGDK
Putative surface antigen 2 (CA-2) PWU84979.1 0.57 DKPSPFGQAAGGEKPPPFGPAAAGDK
12 Putative trans-sialidase PWU87189.1 89.89 MPAGTSEEGSRGDNSMPAGASEEGSRG
Putative surface antigen 2 (CA-2) PWU84979.1 6.84 DKPPPFGQAAAGDKPSLFGQAAAGDKP
R27-2 protein XP_818212.1 3.16 KVAEAEKQRAAEATKVAEAEKQR
Putative trans-sialidase PWU87189.1 0.11 SSMPAGTSQEGIRDDRSMRGGASEEGSRDD
13 Putative surface antigen 2 (CA-2) PWU84979.1 99.97 SPFGQAAAGDKPPPFGQAAAGDKPSPFGQAAAG
Hypothetical protein C4B63_22g72 PWU95420.1 0.02 QEGQAETKRNSVRRGDDPPSAAATPAGTT
Putative surface antigen 2 (CA-2) PWU84979.1 0.01 DKPPPFGQAASGEKPSPFGKAAAGEKPSPF
14 <17.2% in-frame inserts
15 <17.2% in-frame inserts
16 <17.2% in-frame inserts
17 Putative surface antigen 2 (CA-2) PWU84979.1 97.78 AAGDKPSPFGQAAAGDKPPPFGQAAAGDKP
Putative microtubule-associated protein PWU97874.1 1.14 QEDVGPRHVDPDHFRSTTQD
Hypothetical protein XP_803285.1 1.09 SRHWWQCHEWRGTGRAQGLGVEMLVKAKDGGSEPLT
18 <17.2% in-frame inserts
19 Putative surface antigen 2 (CA-2) PWU84979.1 93.81 AAAGDKPSPFGQAAAGDKPSPFG
Putative surface antigen 2 (CA-2) PWU84979.1 6.19 PPFGQAAADDKPPPFGQAAAGEK
20 <17.2% in-frame inserts
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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

A T3 B B13 MAP
2.5 B13 T0 2.0 2.0
Healthy
Lsh **** ****

O.D. (490 nm)

O.D. (490 nm)
2.0 1.5 1.5
Non-Responder
D.O. (490 nm)

1.5 Responder 1.0 1.0

1.0 0.5 0.5

0.5 Healthy Lsh

0.0 0.0
T0 T3 T0 T3
0.0
Criterion 1

48
49
50
51
52
53
54
55
56
57
58
59
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Criterion 2 Hypothetical protein TcG_12368 R27-2 protein

48
49
50
51
52
53
54
55
56
57
58
59
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Patient 2.0 1.5

O.D. (490 nm)

O.D. (490 nm)
2.0 MAP 1.5
**** 1.0
****
1.0
1.5
D.O. (490 nm)

0.5
0.5
1.0
0.0 0.0
T0 T3 T0 T3
0.5 Healthy Lsh

0.0
Criterion 1
100
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
Criterion 2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
Patient
C

Relative reactivity (%)

Orthos Vitros
2.0 Hyp TcG_12368 Hyp TcG_12368
B13
1.5
50
MAP
D.O. (490 nm)

R27-2
1.0

0.5
Healthy Lsh
0
0.0 T0 60d 6m 12m T3
Criterion 1 Time
48
49
50
51
52
53
54
55
56
57
58
59
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47

Criterion 2
48
49
50
51
52
53
54
55
56
57
58
59
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47

Patient

1.5 R27-2
D Criterion 1 Criterion 2
D.O. (490 nm)

1.0 20 20
Parasite load (p/mL)

Parasite load (p/mL)

15 15 R
0.5 Healthy Lsh NR
10 10

0.0 5 5
Criterion 1
48
49
50
51
52
53
54
55
56
57
58
59
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47

Criterion 2
48
49
50
51
52
53
54
55
56
57
58
59
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47

Patient
0 0
T0 60d 6m 12m T3 T0 60d 6m 12m T3
Time Time

Fig 5. Antigen validation. (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).
https://doi.org/10.1371/journal.pntd.0011019.g005

identified, one should be cautious when analyzing the somewhat small list of antigens in our
study. A more complete list of antigens and epitopes recognized by patients in each stage of
Chagas disease can be found in our original study [13].
Although the gPhage selection strategy seems to have favored the selection of major immu-
nodominant epitopes, such as CA-2, Mucin and MAP, we did observe an intriguing trend.
Overall, sera obtained from patients before treatment reacted with a larger diversity of antigens
(Fig 4B); after benznidazole treatment, the great majority of patients reacted preferentially
with the CA-2 antigen, including one patient that switched from reactivity with the MAP anti-
gen to CA-2 (patient #11; Tables 2 and 3). Unfortunately, the small number of patients in the
cohort limited further association analyses between specific antigens or epitopes versus
response to treatment, disease status or clinical evolution. Future studies performed with a
larger cohort of patients, with and without the pre-clearing step to favor the identification of

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011019 January 6, 2023 17 / 20

PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

minor antigens/epitopes as well, will be necessary to further evaluate the power of gPhage as a
diagnostic tool. In sum, gPhage may prove to be an important differential for developing new
diagnostic tools and for the discovery of novel markers relevant for cure of Chagas disease.

Supporting information
S1 Table. Complete clinical and diagnostic data for patients used in phage display pan-
ning.
(XLSX)
S2 Table. Summary of bioinformatic analyses for patients at T0.
(XLSX)
S3 Table. Summary of bioinformatic analyses for patients at T3.
(XLSX)
S4 Table. Summary of antigenic clusters for selected peptides.
(XLSX)
S1 Data. Clusters of antigens for individual patients.
(RAR)
S1 Fig. Antigen validation (individual patients) and Orthos Vitros assay.
(PDF)

Acknowledgments
We acknowledge technical assistance provided by Dr. Layla Martins regarding Next Genera-
tion Sequencing and Célia Lúdio Braga with supply of materials.

Author Contributions
Conceptualization: Luis Antonio Rodriguez Carnero, Edécio Cunha-Neto, Ester Cerdeira
Sabino, Ricardo José Giordano.
Data curation: Luis Antonio Rodriguez Carnero, João Carlos Setubal, Edécio Cunha-Neto,
Ester Cerdeira Sabino, Ricardo José Giordano.
Formal analysis: Luis Antonio Rodriguez Carnero, Andréia Kuramoto, Léa Campos de Oli-
veira, Jhonatas Sirino Monteiro, João Carlos Setubal, Edécio Cunha-Neto, Ester Cerdeira
Sabino, Ricardo José Giordano.
Funding acquisition: Ester Cerdeira Sabino, Ricardo José Giordano.
Investigation: Luis Antonio Rodriguez Carnero.
Methodology: Léa Campos de Oliveira, Jhonatas Sirino Monteiro, João Carlos Setubal, Ester
Cerdeira Sabino, Ricardo José Giordano.
Project administration: Ricardo José Giordano.
Resources: Andréia Kuramoto, Léa Campos de Oliveira, Jhonatas Sirino Monteiro, João Car-
los Setubal, Edécio Cunha-Neto, Ester Cerdeira Sabino, Ricardo José Giordano.
Software: Jhonatas Sirino Monteiro, João Carlos Setubal.
Supervision: Edécio Cunha-Neto, Ester Cerdeira Sabino, Ricardo José Giordano.

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PLOS NEGLECTED TROPICAL DISEASES gPhage predicts benznidazole treatment response in Chagas disease

Validation: Andréia Kuramoto, Léa Campos de Oliveira, Ester Cerdeira Sabino, Ricardo José
Giordano.
Writing – original draft: Luis Antonio Rodriguez Carnero, Edécio Cunha-Neto, Ester Cer-
deira Sabino, Ricardo José Giordano.
Writing – review & editing: Luis Antonio Rodriguez Carnero, João Carlos Setubal, Edécio
Cunha-Neto, Ester Cerdeira Sabino, Ricardo José Giordano.

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