Appl Microbiol Biotechnol

DOI 10.1007/s00253-014-5968-0

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Phage-displayed peptides as capture antigens in an innovative

assay for Taenia saginata-infected cattle
Rafaela L. Fogaa & Janana Capelli-Peixoto & Isabel B. Yamanaka &
Rodrigo P. M. de Almeida & Joo Carlos D. Muzzi & Mariangela Borges &
Alvimar J. Costa & Carlos Chvez-Olortegui & Vanete Thomaz-Soccol &
Larissa M. Alvarenga & Juliana de Moura

Received: 5 May 2014 / Revised: 16 July 2014 / Accepted: 18 July 2014

# Springer-Verlag Berlin Heidelberg 2014

Abstract Bovine cysticercosis is detected during the routine antibody binding. Tsag 1 (HFYQITWLPNTFPAR), the most
post mortem examination of carcasses by visual inspection f r e q u e n t a f f i n i t y - s e l e c t e d c l o n e , a n d Ts a g 6
(knife and eye method). However, the sensitivity of this pro- (YRWPSTPSASRQATL) shared similarity with highly con-
cedure is several times lower than immunoassays, even when served proteins from the Taeniidae family with known immu-
it is performed by qualified professionals. In the present study, nogenicity. Due to their epitopic or mimotopic properties,
a new generation capture antigens were screened from a phage these affinity-selected phages could contribute to the rational
display peptide library using antibodies from Taenia saginata- design of an ante mortem immunodiagnosis method for bo-
infected animals. Eight phage clones were selected, and one, vine cysticercosis, as well as an epitope-based vaccine to
Tsag 3 (VHTSIRPRCQPRAITPR), produced similar results interrupt the taeniosis/cysticercosis complex.
to the T. saginata metacestode crude antigen (TsCa) when
used as a capture antigen in an ELISA. The phage-displayed Keywords Bovine cysticercosis . Immunodiagnosis .
peptides competed with TsCa for binding sites, reducing the Phage-displayed peptide . Taenia saginata
reactivity by approximately 30 %. Alanine scanning indicated
that proline, arginine, and serine are important residues for
Introduction
Rafaela L. Fogaa and Janana Capelli-Peixoto contributed equally to this
work. Taenia saginata is a tapeworm responsible for zoonosis whose
R. L. Fogaa : J. Capelli-Peixoto : I. B. Yamanaka : biological cycle is characterized by the taeniosis/cysticercosis
R. P. M. de Almeida : J. C. D. Muzzi : M. Borges : complex. The complex occurs most commonly in environ-
L. M. Alvarenga : J. de Moura ments that lack sanitation and/or effective methods of meat
Departamento de Patologia Bsica, UFPR, Curitiba, PR, Brazil
inspection and control. This parasitosis is found in both
R. L. Fogaa : V. Thomaz-Soccol : L. M. Alvarenga : J. de Moura developing (Thomaz-Soccol et al. 2010) and developed
Ps-graduao em Engenharia de Bioprocessos e Biotecnologia, countries (Dorny and Praet 2007; Allepuz et al. 2012;
UFPR, Curitiba, PR, Brazil Calvo-Artavia et al. 2013).
J. Capelli-Peixoto : I. B. Yamanaka : M. Borges :
The adult parasite is found in the small intestine of humans
V. Thomaz-Soccol : L. M. Alvarenga : J. de Moura (*) (taeniosis), the only definitive host. The life cycle involves the
Ps graduao em Microbiologia, Parasitologia e Patologia, UFPR, infection of cattle, which is the intermediate host, via the
Curitiba, PR, Brazil ingestion of T. saginata eggs scattered in pasture or water,
e-mail: [email protected]
culminating in the development of the infective larval stage
A. J. Costa (metacestode) in the musculature (cysticercosis). Human in-
CPPARCentro de Pesquisas em Sanidade Animal, UNESP, fection occurs by eating raw or unproperly cooked bovine
Jaboticabal, SP, Brazil meat that contains the viable larval stage (Hoberg 2002).
The development of the metacestode in the bovine muscu-
C. Chvez-Olortegui
Departamento de Bioqumica e Imunologia, UFMG, Belo Horizonte, lature rarely causes symptoms, but it causes financial losses to
Brazil the livestock sector because of the condemnation of infected
 Appl Microbiol Biotechnol

animals. The requirement for freezing lightly infected cattle centrifugation (7,500g, 5 min, 4 C), the protein concentra-
carcasses to inactivate T. saginata cysticerci devalues the meat tion in the supernatant was determined according to the meth-
and increases the costs by extra handling. In addition, the od described by Bradford (1976) and the samples were stored
presence of cysts in the meat is one of the ways that facilitates at 20 C.
the continuity of the taeniosis/cysticercosis complex, which
makes taeniosis an important social and public health Anti-T. saginata metacestode sera
problem.
Bovine cysticercosis is identified during routine post Sera from 1,075 animals were subjected to an indirect ELISA
mortem inspections by visual detection in some classical using the TsCa. In brief, microtiter plates were coated over-
locations such as heart, tongue, masseter, diaphragm, shoul- night at 4 C with 10 g/mL of TsCa diluted in 0.05 M
der, and intercostal muscles; although they can also be found carbonatebicarbonate buffer (pH 9.6). After washing
in kidney, lungs, liver, and anterior and posterior muscles, (0.05 % Tween 20 in saline) and blocking (0.05 M PBS,
some studies have shown that the cysticerci apparently have pH 7.4, and 2 % casein) for 1 h at 37 C, the serum was
no preferred sites (Minozzo et al. 2002). diluted to 1:100 with incubation buffer (0.25 % casein, 0.05 %
Some studies indicate that the post mortem inspection Tween 20, PBS) and added to the wells. After incubation for
procedure has low sensitivity, even when it is performed by 1 h at 37 C and washing, horseradish peroxidase (HRP)-
qualified professionals, i.e. it may fail to detect cysts, partic- labeled rabbit anti-bovine immunoglobulin G (IgG; 1:7,500)
ularly when carcass infection is mild. Thus, the current pro- diluted in incubation buffer was added to the wells. The
cedure is not completely effective in estimating the prevalence reaction was visualized using OPD solution (0.2 mg/mL in
of bovine cysticercosis (Dorny et al. 2000). 0.5 M citrate buffer, pH 5.2, and 0.02 % hydrogen peroxide)
Immunoassays have been proposed as an alternative ap- and then stopped with 20 L of 2 N sulfuric acid, and the
proach for identifying infected cattle because they have higher absorbance was measured at 490 nm. The cutoff value was
sensitivity than visual inspections (Dorny et al. 2000; calculated as the mean of five negative sera plus three standard
Minozzo et al. 2004; Allepuz et al. 2012; Eichenberger et al. deviations. Each negative serum was obtained from an animal
2013). whose carcass was rigorously analyzed.
Based on our previous studies on the identification of B cell
epitopes and their utilization in the serum diagnosis of human Specific Ig preparation
cysticercosis (Hell et al. 2009) and vaccine (Capelli-Peixoto
et al. 2011), in the present investigation, phage display tech- Serum-derived specific Igs were obtained according to the
nology (Smith 1985; Bonnycastle et al. 1996) was used to procedures described by Hell et al. (2009), with some modi-
screen epitopes recognized by antibodies from bovines infect- fications. The total IgG from 120 serologically positive cattle
ed with T. saginata metacestodes to develop a new generation with high titers was purified according to the procedure
of antigens. (Minozzo et al. 2004) based on the reactivity against TsCa;
Antibodies against the metacestode crude antigen were 1 mg TsCa was electrophoresed, transferred onto a nitrocellu-
used for immunopanning phage-displayed libraries. The lose membrane, and blocked (PBS, 0.3 % Tween 20). Then,
screened molecules were capable of distinguishing between the membrane was incubated overnight at 4 C with 1 mg of
positive and negative sera, and they could compete with the total IgG diluted in PBS containing 0.05 % Tween 20 (PBS-
native antigen for binding to the parent antibody, suggesting T). After intensive washing, the bound specific IgG was eluted
the potential usefulness of these peptides as capture antigens with 0.1 M glycine and 0.15 M NaCl (pH 2.8) and neutralized
for the ante mortem identification of T. saginata-infected with Tris/HCl 1 M (pH 9.0). The specific IgG concentration
cattle. was determined by measuring absorbance at 280 nm after
dialysis against PBS.

Material and methods Immunoscreening of phage display libraries

Antigen preparation Five biopanning rounds were performed by incubating four

phage display random libraries kindly provided by Dr. J. Scott
Viable T. saginata metacestodes were collected and dissected (Simon Fraser University, Burnaby, Canada), which express
from the bovine skeletal muscle. After removing the outer 8-mer (LX4), 15-mer (X15), 12-mer (XCX8CX), and 17-mer
capsule, the scolices were ground in a mortar containing 2 mL (X8CX8) peptides (Bonnycastle et al. 1996) with specific
cold phosphate-buffered saline (PBS, pH 7.4) and protease bovine IgG. During the first panning, an immunotube
inhibitor cocktail (Fermentas) and then subjected to five cy- (Nunc) was coated overnight with 5 g/mL of anti-TsCa
cles of sonication (40 Hz, 10 s) with 1-min rest on ice. After IgG diluted in 1.5 mL carbonate buffer (100 mM NaHCO3,
Appl Microbiol Biotechnol

pH 9.6) at 4 C. In the second panning, 1 g/mL of anti-TsCa In a similar way to the procedure described above, 24
IgG was used for coating, with 0.5 g/mL in the subsequent bovine sera were tested against 10 g/mL of TsCa and
pannings. After washing with TBS-T (50 mM Tris, 150 mM 1010 TU/mL for each of the eight phage-displayed mimotopes
NaCl, pH 7.5, and 0.05 % Tween 20) and blocking [TBS-T (Tsag 1Tsag 8) on the ELISA plate to verify whether the use
and 3 % bovine serum albumin (BSA) 3 %], 11011 or 1 of these phage-derived peptides could obtain results similar to
1012 transducing units (TU) of each library were diluted in those produced using TsCa as antigen.
1.5 mL of TBS-T and added to the immunotube, which was
incubated overnight at 4 C. The unbound phages were re- Competition assays
moved by intensive washing. Affinity-selected phage-
displayed peptides were obtained by competition assay after In the first competition ELISA, the microtitration plate was
incubation at room temperature for 90 min with TsCa at a coated overnight at 4 C with TsCa at a concentration of
concentration 100 times higher than that of bovine IgG. The 10 g/mL. At the same time, 1011 TU/mL of each phage,
eluted phages were cloned after infecting Escherichia coli including the wild-type phage or a phage pool, were incubated
K91 cells (Bonnycastle et al. 1996). overnight at 4 C with bovine serum at a dilution of 1:600.
After blocking and washing, a solution of phages plus serum
Biopanning enrichment analysis and screening was added to the coated plate.
of phage-displayed peptides In the second assay, the microtitration plate was coated with
each of the different phages, including the wild-type phage or a
To analyze the enrichment of pannings, a 96-well microtiter plate pool phage, at a concentration of 1011 TU/mL. Bovine serum at a
was coated with 10 g/mL anti-TsCa IgG, washed, and blocked, dilution of 1:600 was incubated overnight at 4 C with TsCa at
as described above. The bacterial supernatant containing phage three different concentrations 10, 100, and 500 g/mL. After
particles from each panning, wild-type phage (negative control) blocking and washing, the solution of TsCa plus bovine serum
diluted at 1:2 in incubation buffer, or 1011 TU/mL of individual was added to the coated plate. In both assays, antibody binding
phage clones was added to each well. After incubation for 2 h at was determined by incubation with peroxidase-conjugated rabbit
37 C and washing, the reaction was detected by adding anti-bovine IgG (1:7,500). The absorbance values were measured
peroxidase-conjugated anti-M13 antibody IgG. The reactivity at 490 nm, and all of the analyses were performed in triplicate.
of clones was determined for selection purposes. Only those
clones showing a reactivity value twofold higher than the nega- SPOT synthesis and alanine scanning
tive control were selected for further sequencing using the primer
5-TCG GCA AGC TCT TTT AGG-3. Based on ELISA reactivity, the synthetic peptides correspond-
ing to three phage-displayed peptides and their analogs with
Sequence analysis of the phage-displayed peptides alanine replacement (alanine scanning) were prepared on
membranes using the SPOT technique, as previously de-
The translated amino acid sequences were analyzed using scribed (Laune et al. 2002). In peptides with alanine in their
UniProt (UniProt Consortium 2014) and GenBank (Benson original sequence, this residue was substituted by serine.
et al. 2013) databases. Matches of the peptide sequences with Nonspecific binding sites were blocked by incubation over-
Taenia sp. antigens (Manoutcharian et al. 1996; Rassy et al. night at 4 C with 0.1 % TBS-T and 3 % BSA. The membrane
2010) were examined using the Dialign-TX program was probed with a pool of negative or positive bovine serum
(Subramanian et al. 2008). (diluted to 1:3,000) for 90 min at room temperature. After
washing, antibody binding was determined by incubation for
Indirect ELISA for the determination of anti-peptide 1 h at room temperature with peroxidase-conjugated rabbit anti-
antibodies bovine IgG (1:30,000) and by ECL detection. The signal inten-
sities (ImageJ) were obtained based on comparisons with spot
To evaluate the antigenicity of phage-displayed peptides, an with the highest pixel value, which was considered to be 100 %.
indirect ELISA was conducted according to the procedure
described above. Microtitration plates were coated overnight
at 4 C with 1011, 1010, 109, 108, and 107 TU/mL of phage- Results
displayed peptides, wild-type phage, or 10 g/mL of TsCa.
After blocking and washing, positive and negative sera (dilut- Immunoselection of positive serum for cysticercosis
ed to 1:100) were added. Antibody binding was determined by by indirect ELISA
incubation with peroxidase-conjugated rabbit anti-bovine IgG
(1:7,500). The absorbance values were measured at 490 nm. Out of a total of the 1,057 bovine sera tested against TsCa, 120
All of the analyses were performed in triplicate. were selected based on their titers according to ELISA. The
 Appl Microbiol Biotechnol

sera that were considered positive had absorbance measure- Table 1 Prevalence, sequence, and most frequent residues in the phage-
displayed peptides
ments that were at least threefold higher than the cutoff.
Clone Amino acid Prevalence Sequence
residues (n)
Enrichment of anti-TsCa antibody-binding phages
Tsag 1 15 15/24 HFYQITWLPNTFPAR
and reactivity analysis
Tsag 2 12 1/24 TCIWQWPDWACK
Tsag 3 17 1/24 VHTSIRPRCQPRAITPR
Figure 1 illustrates the enrichment of anti-TsCa antibody-
Tsag 4 17 1/24 MGIRALPPCQNARQRLS
binding phages after five rounds of panning (PI, PII, PIII,
Tsag 5 15 3/24 LSTHTKLNKADIQMP
PIV, and PV) and the reactivity of each pool against anti-
Tsag 6 15 1/24 YRWPSTPSASRQATL
TsCa antibodies after subtracting the background signal,
which was obtained using the wild-type phage. The phage Tsag 7 15 1/24 GAARHCQPASPATMM
titer was reduced between the fourth and fifth rounds, but the Tsag 8 12 1/24 FCMSTCSGLKCQ
reactivity of the phage eluted after the fifth panning was three
orders greater than that from the fourth.
Comparison between the antigenicity of TsCa
and phage-displayed peptides
Prevalence of T. saginata mimotope sequences
Given that there is no gold standard serological assay for
Twenty-four phages that harbored peptides with higher anti- bovine cysticercosis, each phage-displayed peptide was com-
genicity (Tsag 1Tsag 8) were selected and sequenced. Only pared with TsCa in relation to their antigenicity, i.e. to assure if
12-, 15-, and 17-mer library peptides were detected in the the selected peptide could be employed as efficiently as the
selected phages (Table 1), Tsag 1 (15/24), Tsag 5 (3/24) being T. saginata crude antigen in ELISA to identify infected cattle.
the most prevalent. Accordingly, 24 bovine sera were tested, and 19 were positive
As expected, paired cysteines that could form disulfide (Fig. 2a, b, and c), whereas five sera were considered negative
bridges were found only in the 12-mer sequences (Tsag 2 using TsCa as the antigen (Fig. 2d).
and Tsag 8) from a disulfide-constrained peptide library. Tsag 1 was the most prevalent phage-displayed peptide
The mimotopes did not show any consensus sequences, but selected, but it only identified 11 positive [11/19 (Fig. 2a, b,
the three most common amino acid residues (proline, n=13; and c)] and four negative sera [4/5 (Fig. 2d)]. Similar results
alanine, n=12; threonine, n=11) were present in seven out of were obtained with Tsag 4 and Tsag 6, although they had
eight phage-displayed peptides. Arginine (n=11) and gluta- lower cutoff values.
mine (n=9) were also present in five and eight peptides, Although Tsag 5 was the second most prevalent phage-
respectively. derived peptide, it produced the most discordant results with
respect to negative sera, i.e. four sera that were considered to
be negative using TsCa yielded positive results with Tsag 5
(Fig. 2d). In contrast, the results obtained using Tsag 2 and
Tsag 7 exhibited 100 % agreement with those using TsCa with
respect to the negative sera, but they were unable to identify
most of the positive sera.
Better agreement with TsCa was obtained when Tsag 3 was
used as antigen, which was able to detect 15/19 positive sera
(Fig. 2a, b, and c) and 4/5 negative sera (Fig. 2d).

Competition assays

Fig. 1 Enrichment and reactivity of phage-displayed peptides selected

The majority of phage-displayed mimotopes including the
using antibodies against TsCa after five rounds. The number of phage phage pool were able to compete for the antibodies after
particles recovered after each panning (PI, PII, PIII, PIV, and PV) and previous incubation reducing TsCa reactivity by an average
their reactivity against sera from T. saginata-infected cattle are shown as of 30 %. Tsag 2, Tsag 4, and Tsag 5 were not able to impair the
transducing units (TU) and the absorbance (490 nm), respectively. TsCa
was used as control. The mean absorbance of the wild-type phage (three
TsCa-antibody interaction in the same manner as the others,
replicate assays) was subtracted from the absorbance of each panning while they decreased reactivity by 26, 22, and 28 % respec-
assays for expressing the absorbance values tively. As expected, the wild-type phage did not have an effect
Appl Microbiol Biotechnol

Fig. 2 Reactivity of selected phage clones and TsCa with bovine sera. TsCa reactivity. Each symbol indicates a serum assayed against each
Twenty-four bovine sera were evaluated by ELISA using TsCa or each phage-displayed peptide or TsCa. The solid line indicates the cutoff value,
phage clone as capture antigens. Nineteen samples were classified as i.e. mean of the absorbance values for definite negative sera plus three
positive (a, b, c) and five as negative (d) for cysticercosis based on their standard deviations

on the interaction between bovine antibodies and TsCa Among the three peptides tested, only Tsag 2
(Fig. 3a). (TCIWQWPDWACK) failed to exhibit reactivity with bovine
However, when the bovine serum was previously incubat- sera positive for cysticercosis (Fig. 4b). By contrast, the alanine
ed with TsCa at different concentrations (10, 100, and 500 g/ scanning of the peptides Tsag 1 (HFYQITWLPNTFPAR) and
mL) and assayed against the phage-displayed peptides, there Tsag 3 (VHTSIRPRCQPRAITPR) resulted in the decreased
was a little reduction in the absorbance compared with that in antibody recognition. In Tsag 1, the replacement of
the assay without the competitor (Fig. 3b). arginine in position 15 (R15) by alanine (A) resulted
in about 80 % reduction of binding, while the substitu-
tion of phenylalanine (F2) and prolines (P9 and P13) by
Alanine scanning alanine decreased the reactivity by at least half
(Fig. 4c).
To characterize the interactions between the phage-displayed For Tsag 3 (Fig. 4d), the substitution of A13 by
mimotopes and the T. saginata-positive bovine serum, three serine slightly increased the reactivity while the replace-
peptides and a derived series of alanine analogs were assayed ment of S4 and I14 by alanine interfered with peptide-
for sera reactivity as illustrated in the Fig. 4a. antibody interactions compared with the unmodified
Tsag 1 and 3 were selected from the 15- and 17-mer peptide. As observed with Tsag 1, this experiment indi-
libraries because they were the most prevalent in the phage cated the important roles of some proline (P11 and P16)
display selections and they obtained the best agreement with and arginine (R6 and R17) residues, whose absence
TsCa in ELISA, respectively. The phage-derived peptide Tsag decreased the reactivity to bovine antibodies between
2 was also selected as representative of the 12-mer library. 42 and 66 %.
 Appl Microbiol Biotechnol

Fig. 3 Phage competitive

ELISA. a High titer bovine serum
was pre-incubated with 1011 TU/
mL wild-type phage (WT) or
phage clones and tested on the
TsCa-coated plates (10 g/mL).
The reactivity of the high titer
bovine serum against TsCa was
considered 100 % and used as
reference. b High titer serum was
pre-incubated with TsCa (10, 100,
or 500 g/mL) and assayed on
phage-coated plates (1011 TU/
mL). In both experiments, the
reaction was detected using a
HRP-coupled anti-bovine
antibody

Discussion Thus, specific antibodies were selected based on their

T. saginata metacestode crude antigen (TsCa) reactivity and
Bovine cysticercosis is a zoonosis that has major impact in used in five pannings against four phage display random
underdeveloped and developing countries because it causes peptide libraries (Bonnycastle et al. 1996). Using competitive
economic losses in livestock due to refrigeration, devaluation, elution, 24 clones were screened to obtain eight phage-
and condemnation of infected carcasses, as well as the effects displayed peptides.
of the taeniosis/cysticercosis complex. Peptide antigenicity was compared with that of TsCa
The knife and eye method of meat inspection is currently using ELISA, and some peptides appeared to be better
used to verify the presence of cysticerci in cattle carcasses, but capture antigens than others. Using Tsag 3, both anti-
this procedure is not sensitive. Immunological techniques gens were in agreement in 80 % of cases. Curiously,
have been proposed as a powerful alternative for detecting some phages such as Tsag 2 and Tsag 7 were unable to
infected animals instead of visual inspections (Dorny et al. identify most of the positive sera, although the same
2000; Minozzo et al. 2004; Allepuz et al. 2012). phage selection protocol was used.
Biotechnological strategies, such as the phage display tech- When the phage pool was used as a competitor antigen in
nique, are recognized as valuable methods for selecting and relation to TsCa, the reactivity impairment of bovine antibod-
identifying peptide ligands for antibodies. Phage-displayed ies and TsCa remained the same as the average, which was
peptides have shown to be excellent capture antigens in assays probably because of the polyclonal character of the bovine
for parasitic diseases, including human neurocysticercosis sera. Although the reduction in the absorbance values did not
(Hell et al. 2009; da Silva Ribeiro et al. 2010; Gazarian et al. exceed 35 %, this percentage indicates good affinity between
2012), and they could provide an alternative for the serolog- the ligands and the bovine antibodies, particularly if these
ical diagnosis of bovine cysticercosis. results are compared with studies using monoclonal
Appl Microbiol Biotechnol

Fig. 4 Alanine scanning of three linear peptides selected by phage indicated with asterisk. Quantitative analysis of SPOT reactivity of c Tsag
display on a SPOT membrane. a Schematic representation of reaction 1 and d Tsag 3. Each bar represents the reactivity of a peptide whose
between the bovine antibody and the wild-type peptide and their analogs sequence contains alanine or serine replacing the indicated amino acid.
with alanine replacement (alanine scanning) on a SPOT membrane. b The reference peptide (first bar) was used as control. SPOT intensities
Antigenic reactivity of cellulose-bound phage-displayed peptides (Tsag 1, were measured using ImageJ
2, and 3) using a positive bovine serum pool. The wild-type peptide is

antibodies whose phage-displayed peptides inhibited binding for recognition by antibodies, and this characteristic is pre-
by 50 % (Guo et al. 2010). served only when it is exposed on the surface of the phage
Another evidence of this strong affinity was observed when (Martin et al. 2002).
the TsCa was not able to significantly interfere with the Regarding the homology between the phage-displayed pep-
reactivity between the bovine antibodies and the phage- tides and Taenia sp. proteins, only two shared significant
displayed peptides, even at high concentrations (100 and similarity with already described proteins. Tsag 1
500 g/mL). (HFYQITWLPNTFPAR) shared a core region with a highly
Although no consensus sequences were verified, the eight conserved sequence from GK-1 (GYYYPSDPNTFYAPPYQ)
clones shared some common characteristics. The most fre- (Rassy et al. 2010), a peptide from KETc7, which is a proline-
quent amino acids in the peptides selected were proline, rich protein found in the Taeniidae family (Manoutcharian
threonine, arginine, and serine. Similarly, previous screenings et al. 1996). This protein induces protection against cysticer-
of the mimotopes of T. solium, a platyhelminth that is phylo- cosis (Toledo et al. 1999) and has reasonable capacity for
genetically related to T. saginata, identified peptides whose identifying neurocysticercosis patients (Gevorkian et al.
sequences also contained proline, arginine, and serine as the 1996). The Tsag 6 sequence (YRWPSTPSASRQATL) shared
most common residues (Manoutcharian et al. 1999). similarities with another conserved region in the protein
In the present study, the importance of proline in some KETc1 (PxSTPxA) from cestodes (Rassy et al. 2010) includ-
sequences was confirmed by alanine scanning assay using ing T. saginata (PTSTPLATLAR). The peptides GK1 and
SPOT synthesis membranes. After replacing proline by ala- KETc1 are both components of S3Pvac, which is an anti-
nine residue in Tsag 1, which is the most frequent affinity- cysticercosis vaccine that induces a porcine immunological
selected clone, there was a significant loss in its reactivity to response against T. solium infection and reduces parasite load
bovine sera. In the case of Tsag 3 whose ELISA results were (Sciutto et al. 2013).
also very similar to the crude antigen, the reactivity was The results of the present study indicate the potential ap-
decreased when the proline, arginine, and serine residues were plication of screened peptides as capture antigens in an im-
changed, demonstrating that the presence of these amino acids munoassay for bovine cysticercosis. Additional studies using
is important for antibody binding. these peptide sequences are required, but their epitopic or
No evidence of reactivity was verified with Tsag 2. The mimotopic properties could also facilitate the rational design
conformation of this displayed peptide is probably essential of an epitope-based vaccine to prevent infection by
 Appl Microbiol Biotechnol

T. saginata metacestodes, as well as allowing better under- Hell RC, Amim P, de Andrade HM, de Avila RA, Felicori L, Oliveira AG,
Oliveira CA, Nascimento E, Tavares CA, Granier C, Chvez-
standing of the immunogenic proteins that are mimetized by
Olrtegui C (2009) Immunodiagnosis of human neurocysticercosis
these peptides. using a synthetic peptide selected by phage-display. Clin Immunol.
doi:10.1016/j.clim.2008.10.012
Acknowledgments This research was supported by Fundao Arau- Hoberg EP (2002) Taenia tapeworms: their biology, evolution and socio-
cria and CNPq. We thank Ronaldo Nakato and Fernanda C. Cancelli economic significance. Microbes Infect. doi:10.1016/S1286-
Vieira for their skillful technical contributions. We also thank Dr. J. Scott 4579(02)01606-4
for the phage libraries and Dr. Valmir Kowaleski Souza and Antnio Laune D, Molina F, Ferrires G, Villard S, Bs C, Rieunier F, Chards T,
Carlos Nunes for the T. saginata cysticerci. Granier C (2002) Application of the Spot method to the identifica-
tion of peptides and amino acids the antibody paratope that contrib-
ute to antigen binding. J Immunol Methods. doi:10.1016/S0022-
1759(02)00140-0
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