Physicochemical and immunological characterization of chitosan-coated bacteriophage nanoparticles for in vivo mycotoxin modeling

Carbohydrate Polymers 185 (2018) 63–72

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Physicochemical and immunological characterization of chitosan-coated
bacteriophage nanoparticles for in vivo mycotoxin modeling

T

Carla Yoko Tanikawa de Andradea, Isabel Yamanakaa, Laís S. Schlichtab, Sabrina Karim Silvaa,
⁎
Guilherme F. Pichethb, Luiz Felipe Carona, Juliana de Mouraa, Rilton Alves de Freitasb, ,
⁎
Larissa Magalhães Alvarengaa,
a
b

Limq, Basic Patology Department, Federal University of Paraná, 81530-900 Curitiba, PR, Brazil
Biopol, Chemistry Department, Federal University of Paraná, 81531-980 Curitiba, PR, Brazil

A R T I C L E I N F O

A B S T R A C T

Keywords:
Phage display
Mimotope
Aflatoxin B1

Peptide carrier
Mucosal vaccine
Chitosan

To propose a novel modeling of aflatoxin immunization and surrogate toxin conjugate from AFB1 vaccines, an
immunogen based on the mimotope, (i.e. a peptide-displayed phage that mimics aflatoxins epitope without toxin
hazards) was designed. The recombinant phage 3P30 was identified by phage display technology and exhibited
the ability to bind, dose dependent, specifically to its cognate target − anti-AFB1 antibody. In immunization
assay, the phage-displayed mimotope and its peptide chemically synthesized were able to induce specific antiAFB1 antibodies, indicating the proof of concept for aflatoxin mimicry. Furthermore, the phage 3P30 was
homogeneously coated with chitosan, which also provided a tridimensional matrix network for mucosal delivery. After intranasal immunization, chitosan coated phages improved specific immunogenicity compared to
the free antigen. It can be concluded that affinity-selected phage may contribute to the rational design of epitope-based vaccines in a prospectus for the control of aflatoxins and possibly other mycotoxins, and that chitosan
coating improved the vectorization of the vaccine by the mucosal route.

1. Introduction
Aflatoxins are secondary metabolites produced mainly by two
Aspergillus species, namely A. flavus and A. parasiticus (WHO, 2002).
These non-proteinaceous toxins responsible for aflatoxicosis, a disease
which may affect both humans and animals, cause severe liver intoxication, usually leading to hemorrhagic necrosis of the organ, bile
duct proliferation and edema (Wild, Miller, & Groopman, 2015).
The main route of exposure to aflatoxins is through the diet by the
ingestion of aflatoxin-contaminated maize, peanuts (groundnuts), oil
seeds, and tree nuts (Gibb et al., 2015). Although more than 20 aflatoxins have been identified, aflatoxin B1 (AFB1) is the most toxic and
generally present in the largest quantity (Liu & Wu, 2010). AFB1 is also
associated with the development of hepatocellular carcinoma, being
classified since 1993 as group I human carcinogen by the International
Agency for Research on Cancer (WHO, 1993).
Notably, 4.5 billion people from developing countries are chronically exposed to high amounts of aflatoxins and the intake of such
toxins over a long period of time, even at low concentrations, significantly increases the risk of hepatocellular carcinoma and extrahepatic tumors (Gnonlonfin et al., 2013). AFB1 has a wide range of
⁎

biological activities, including genotoxicity, teratogenicity, hepatotoxicity, nephrotoxicity and immunosuppression (Wild et al., 2015).
As animals ingest aflatoxin-contaminated grains, important parameters of production are compromised and attributed to AFB1-induced
tissue damage: highly reactive aflatoxin metabolites (e.g. AFM1) are
formed in animal tissues and, consequently, meat and dairy products
might also represent a potential risk to human health (WHO, 2002,
1993).
The best strategy to avoid aflatoxin intake by the general population
is preventing fungal growth in agricultural products (Wild et al., 2015).
However, when outbreaks occur, any physical or chemical detoxifying
methodologies is able to guarantee complete safety (WHO, 2005; Baek,
Lee, & Choi, 2012). Nonetheless, recent control strategies have been
based on aflatoxin vaccines which perform immune-interception of the
toxin using circulating or site specific antibodies (Wilkinson et al.,
2003; Polonelli et al., 2011 and Giovati et al., 2014). The AFB1-derived
vaccines, however, have been reported to produce a limited immunogenicity likewise such haptens (i.e. small molecule, not antigenic
by itself) may be potentially toxic also when conjugated with protein
carriers.
Therefore, one approach to avoid the toxicity of AFB1 derivatives

Corresponding authors.
E-mail addresses: , (R.A. de Freitas), (L.M. Alvarenga).

/>Received 29 September 2017; Received in revised form 6 December 2017; Accepted 22 December 2017
Available online 28 December 2017
0144-8617/ © 2017 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 185 (2018) 63–72

C.Y.T. de Andrade et al.

Fig. 1. Selection of phage-displayed peptides recognized by anti-AFB1 antibodies. (A) Schematic representation of panning procedure. Phage display libraries were screened against antiAFB1 antibody and after washing unbound phages were removed. Affinity-selected phages were eluted and amplified for next round. (B) Enrichment and reactivity of phage-displayed
peptides selected using antibody against AFB1 after three rounds. The number of phage particles recovered after each panning (PI, PII, and PIII), and their reactivity against anti-AFB1
antibodies are shown as plaque-forming units (pfu) and absorbance (490 nm), respectively. The mean absorbance of the wild type phage (WTP) was subtracted from the absorbance of
each panning assay. (C) Immunological screening of phage clones. Individual colonies containing phages from each panning were amplified and analyzed regarding its binding to specific
antibody by ELISA. Phages were detected using a peroxidase conjugated anti-M13 antibody and reactivity was shown as mean absorbance (490 nm) ± SD. Wild type phage was used as
control.

immunized mice, proving the concept for aflatoxin mimicry.
To develop a mucosa vaccine a chitosan-phage delivery system was
proposed mainly due to several interesting aspects compared to other
routes of drug administration as: large surface area, thin absorption
barrier and low enzymatic metabolic activity (Chang & Chien, 1984,
Yamamoto, Kuno, Sugimoto, Takeuchi, & Kawashima, 2005). Based on
this advantages the selected phage 3P30 was entrapped into a shell of
the chitosan, a well known mucoadhesive biopolymer (Rodrigues,
Dionísio, Remán López, & Grenha, 2012) extensively used as pharmaceutical excipient and as intranasal drug delivery system (Casettari &
Illum, 2014).
Chitosan, a pseudo-natural polymer obtained from chitin, is a linear
polysaccharide composed of D-glucosamine with few amount of Nacetyl-D-glucosamine units bonded via β-(1 → 4) (Jayakumar, Menon,
Manzoor, Nair, & Tamura, 2010). As a bioadhesive material, chitosan is
able to decrease the clearance of formulations from nasal mucosa and to
open the tight junctions in mucosal membranes (Illum, Jabbal-Gill,
Hinchcliffe, Fisher, & Davis, 2001), with no interference in the humoral
immune response after nasal or subcutaneous administration (Illum,
1998). Therefore, mucosal immunization assays revealed that chitosanshelled phages provided a more efficient specific immune response
compared to non-coated phages. In this context, antigen identified as a

relies on the replacement of the toxin by mimotopes, i.e. peptide-displayed phages with potential to mimic the AFB1 molecule. For this
purpose, phage display is the most widely surface display system for the

expression of peptides on the surface of filamentous phage (Galán et al.,
2016). Through phage display, a mimotope selected by the specific
affinity for variable regions of anti-aflatoxin antibodies represents a
potential immunogen to surrogate toxin haptens and provide a more
adequate immunization modeling (Huang, Ru, & Dai, 2011). In fact,
genetically engineered phages present a wide range of applications in
veterinary and medical vaccine research: phage vectors have been used
in vaccines against porcine Circovirus 2 (Gamage, Ellis, & Hayes, 2009),
brown-spider venom toxins (de Moura et al., 2011) and many others
(Sagona, Grigonyte, MacDonand, & Jaramillo, 2016).
In this study, AFB1 mimotopes expressed on a foreign phage surface
were applied as vaccine candidates against aflatoxin. By using a
monoclonal antibody (mAb) against aflatoxin B1 to screen four phage
libraries (Bonnycastle, Mehroke, Rashed, Gong, & Scott, 1996), a peptide mimicking the epitope of AFB1 was successfully isolated and tested
by functional and antigenic assays. The peptide-derived phage that
presented the best results of specificity was selected and evaluated regarding its immunogenic properties. The phage-displayed mimotope
and its synthetic peptide were able to induce humoral response in
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C.Y.T. de Andrade et al.

2.2.2.2. Mimotope characterization (DNA sequencing, bioinformatics and
peptide coupling). The most reactive and specific clones (with an
absorbance at least twice as high compared to WTP) were selected for
DNA sequencing and subsequent identification of the peptide sequence
inserted into the phages (Supplementary data). Peptide sequences of six
valid phage clones 3P8, 3P13, 3P19, 3P20, 3P23, 3P30, irrelevant

phage 3P25 (randomly chosen from screening) and WTP were deduced
using the Expasy server (www.expasy.org) and analyzed with the
HHPred, Pepdraw, PepSearch, PeptideMass and ProtParam programs
to characterize the sequence. The peptide was synthesized and
covalently coupled to protein carriers, as described by Capelli-Peixoto
et al. (2011), in detail in the Supplementary data.

mimotope of a non-proteinaceous molecule may be considered a prospect of an epitope-based vaccine after coating with chitosan.
2. Materials and methods
2.1. Materials
Luria-Bertani broth (LB broth) and LB broth with agar; Tetracycline;
anti-AFB1 monoclonal and polyclonal antibodies; bovine serum albumin (BSA); BSA conjugated with AFB1; Freund’s complete and incomplete adjuvant; O-phenylenediamine dihydrochloride (OPD); acetic
acid; sodium acetate; NaOH; isothiocianate of fluoresceine (FITC) and
all other reagents are PA grade from Sigma-Aldrich. Microtitration
plates (Nunc-Imuno, MaxiSorp F96, Nunc, Roskilde, Denmark); mAb7
monoclonal antibody (Alvarenga et al., 2003); polyclonal antibodies
from non-immunized rabbit or mice (produced by our laboratory Limq);
peroxidase-conjugated anti-M13 antibody produced in mouse (GE
Health Care, Little Chalfont, England); Alexa-fluor 633-conjugated antibody anti-IgG murine (Thermo Scientific, Waltham, USA); coopergrid
coated with a carbon layer (Pelco, Clovis, USA); Uranyl acetate (Polysciences, Warrington, USA). Chitosan was obtained from Purifarma
(São Paulo, Brazil).

2.2.2.3. Immunization
of
mice
with
phage
and
synthetic
peptide. Immunization of mice with phages was performed as

described by Galfrè et al. (1996). Briefly, phage clone 3P30 (1 × 1011
particles) in 100 μL TRIS buffer saline (TBS) was injected
subcutaneously into 3–4-week-old female Swiss mice. Groups of mice
were also injected with BSA conjugated with AFB1 (A6655, SigmaAldrich, USA) or synthetic peptide (25 μg dissolved in 50 mmol L−1
phosphate buffer saline, PBS, 150 mmol L−1 NaCl, pH 7.4). The mice
belonging to the control groups were injected with WTP or with the
irrelevant phage 3P25. All five groups of four mice received adjuvants
(Freund’s complete adjuvant for first immunization, and Freund’s
incomplete adjuvant for subsequent boosters) with phage suspension
(1:1 v/v). Two additional boosters were given at 2–3 week intervals
followed by final injection after one week. All animals were bled seven
days after the fourth injection for serum collection. The sera were kept
at −20 °C until analysis of the immune response elicited by
immunization.

2.2. Methods
2.2.1. Ethics statement
Experimental procedures were performed in accordance with the
institutional guidelines, based on national and international guidelines
(EU Directive 2010/63/EU). Animal procedures were approved by the
Committee on the Ethical Handling of Research Animals from the
Federal University of Paraná (UFPR), Curitiba, Brazil, process number
23075.073175/2015.

2.2.2.4. Indirect ELISA for determination of anti-peptide and anti-AFB1
antibodies. Microtitration plates were coated at 4 °C for 16 h with
10 μg mL−1 of synthetic peptide coupled to BSA, AFB1-BSA or BSA in
carbonate buffer, pH 8.6, as previously described and the reactivity
against serum from mice immunized with peptide conjugated to BSA or
AFB1-BSA was evaluated. After washing, peroxidase-conjugated antiIgG murine antibody (Sigma-Aldrich, USA), diluted 1:4000 in blocking

solution was incubated for 1 h at 37 °C. The specific antibody titer was
derived as the reciprocal sample dilution corresponding to the
OD490nm ≥ 0.05 after correction for BSA reactivity values. The results
were presented as mean titer ± SD per group.

2.2.2. Phage display
The panning procedure (Fig. 1A) was performed as previously described by Scott & Smith (1990) and Lunder, Bratkovic, Urleb, Kreft, &
Strukelj (2008) with some modifications, in detail in the Supplementary
data.
2.2.2.1. Determination of immune reactivity of phage-displayed peptides by
ELISA. To evaluate the antigenicity of phage-displayed peptides, an
indirect ELISA was conducted according to the procedure describe as
follows. Isolated colonies containing phages from each panning were
randomly picked and individually grown for 16 h at 37 °C in LB medium
with 20 μg mL−1 tetracycline. The supernatants containing phages were
obtained by centrifugation (1.6 × 103 g, 20 min, 4 °C) and analyzed
regarding their binding to specific antibody. Microtitration plates
(Nunc-Imuno, MaxiSorp F96, Nunc, Roskilde, Denmark) were coated
for 16 h at 4 °C with cognate targets − anti-AFB1 monoclonal (A9555,
Sigma-Aldrich, USA) or polyclonal antibodies (A8679, Sigma-Aldrich,
USA) − or also with the irrelevant ligands: bovine serum albumin
(BSA), mAb7monoclonal antibody (Lunder et al., 2008) or polyclonal
antibodies from non-immunized rabbit or mice. After washing and
blocking, the following dilutions 1011, 1010, 109, 108 pfu mL−1 of
phage-displayed peptides or wild type phage (WTP) were added and
incubated for 1 h at 37 °C. A wild type phage is identical to phage clones
present in the libraries, but do not express foreign exogenous peptides.
After washing, peroxidase-conjugated anti-M13 antibody produced in
mouse (GE Health Care, Little Chalfont, England), diluted 1:5000 in
blocking solution was incubated for 1 h at 37 °C. Antigen-antibody

complexes were determined by peroxidase activity using Ophenylenediamine dihydrochloride (OPD) (Sigma-Aldrich, USA) as
chromogen and hydrogen peroxide as substrate in citrate buffer (pH
5.0) for measuring the absorbance at 490 nm with Bio-Rad
spectrophotometer (Bio-Rad Laboratories, Berkeley, USA) (Alvarenga
et al., 2003).

2.2.3. Chitosan-coated bacteriophage nanoparticles
2.2.3.1. Encapsulation and characterization of nanoparticles. Chitosan
was obtained from Purifarma (São Paulo, Brazil), and purified as
described by Recillas et al. (2009). All macromolecular chitosan
characterization can be observed in detail in the Supplementary data.
The weight average molar mass (Mw) was determined as
1.1 × 105 g mol−1, the radius of gyration (Rg) was determined as
37 nm and the intrinsic viscosity [η] as 4.0 dL g−1. The degree of
deacetylation (DDA) was determined using two methods:
potentiometric titration (80%) and by 1H NMR (82%) (Supplementary
data).
The phage 3P30 was employed as a substrate for the assembly of
chitosan nanoparticles. The recombinant phage was amplified, titrated
to about 5 × 1013 pfu mL−1 and a final solution was obtained after
dilution in 10 mL ultrapure water to a concentration of
1 × 1011 pfu mL−1.
The phage coating with chitosan was performed using the coacervation/precipitation processes. The first one was attributed to ionic
interaction between chitosan and the negatively phages at pH 4.6.
After, a complex precipitation was performed, using diluted NaOH up
to pH 7.0, inducing the formation of an insoluble shell of chitosan
around the phage. Briefly, the coating was obtained after addition of
10 mL of final solution of phages into 10 mL of chitosan in 0.01 mol L−1
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C.Y.T. de Andrade et al.

acetic acid/sodium acetate buffer, 1 mg mL−1, pH 4.6, under continuous mild stirring. After complete mixture the solution was maintained in agitation for at least 1 h. The solution was then neutralized to
pH 7.0 with a 0.2 mol L−1 NaOH solution, under continuous agitation.
The chitosan coated-bacteriophage were centrifuged at 104 g, 25 °C,
30 min and 1 mL sterile PBS was added to the precipitate.
The efficiency of entrapment (EE) (Eq. (1)) and Loading Capacity
(LC) (Eq. (2)) of bacteriophages into the chitosan shell was determined
by the remained free phages in the supernatant after centrifugation at
pH 7.0 by titration on log-phase Escherichia coli K91.

total phages − free phages ⎞
EE (%) = ⎜⎛
⎟*100
total phages
⎝
⎠

using a micropipette. Six different groups containing four mice were
maintained conscious during the administration on days 1, 14 and 28.
Group 1: Nanoparticles of chitosan-phage 3P30 at 1011 pfu mL−1.
Group 2: Free phage 3P30 at 1011 pfu mL−1. Group 3: A pulse-chase
study was performed to evaluate whether the adjuvant activity might
be observed when chitosan was co-administrated with the phage 3P30
at 1011 pfu mL−1. The interval between the administration of chitosan
and the bacteriophage was 2 h to prevent the phage and chitosan to
interact after administration, anticipating that the cationic chitosan will

be promptly neutralized by the abundantly negatively charged mucins
and/or cleared by mucociliary activity. Group 4: Nanoparticles of
chitosan-WTP at 1011 pfu mL−1. Group 5: Free phage WTP at
1011 pfu mL−1. Group 6: The animals were treated only with chitosan
particles, as a control group.
Blood samples were taken from the orbital plexus on day 35 postadministration and serum samples were maintained at −20 °C prior
ELISA analysis as described above. Microtitration plates were coated
with 10 μg mL−1 of BSA, AFB1-BSA and peptide-BSA as with
1011 pfu mL−1 of the phage 3P30, WTP or irrelevant phage 3P25.
Bronchoalveolar lavages were collected 38 days after the last administration, using a modified procedure described by Vila et al. (2003), in
detail in the Supplementary data.

(1)

total phages − free phages ⎞
LC = ⎜⎛
⎟
⎝ chitosan particle mass ⎠

(2)
12

The total phages was the initial phages added (1,78 × 10 pfu),
and the chitosan particle mass was 0.01 g.
Both uncoated and chitosan-coated bacteriophages were analyzed at
pH 4.6 and 7.0 using dynamic light scattering (DLS) and zeta potential.
The transmission electronic microscopy (TEM) and confocal microscopy
were performed with phages-chitosan at pH 7.0 as described below.
The apparent hydrodynamic diameter of the phages and coated
phages were determined using dynamic light scattering NanoDLS

equipment (Brookhaven, New York, USA). The terminology “apparent”
was used here due to the anisotropy of the phages, meaning that the
values obtained are only used here for comparative purpose. The Zeta
potential was determined for the phages and coated phages with chitosan using a Microtrac Stabino Particle Charge Titration Analyzer
(Particle Metrix GmbH, Meerbusch, Germany). The same condition of
phage concentration was used, as described for DLS measurements.

2.2.3.4. Statistical analysis. The results from various groups were
represented as mean ± standard deviation (SD). Statistical
evaluation was carried out by one way analysis of variance (Anova),
followed by Tukey's post hoc test with the significance level set at
p < 0.05.
3. Results
3.1. Panning-elution selection of isolated mimotopes of AFB1

2.2.3.2. Interaction
of
chitosan-bacteriophage
nanoparticles
by
microscopy. Uncoated-phages
and
coated-phages
were
also
characterized using a chitosan-fluorescein (FITC) prepared as
described by Quemeneur, Rammal, Rinaudo, & Pepin-Donat (2007).
The FITC-chitosan was used to demonstrate the adsorption onto phage
surfaces. To confirm the adsorption, the phage 3P30 was also
previously incubated with a monoclonal antibody anti-phage M13

(27942101, GE Health Care, Little Chalfont, England) for 1 h and
labeled with Alexa-fluor 633-conjugated antibody anti-IgG murine
(Thermo Scientific, Waltham, USA) for 1 h at room temperature
(Supplementary information). The combination of both images in
Green (FITC-Chitosan) and Red (Alexa-fluor) could be observed in
yellow. After the encapsulation process, chitosan-phage nanoparticles
were imaged in a laser scanning confocal multiphoton microscope,
model A1 MP+ (NIKON Instruments Inc., Tokyo, Japan), using a 40X
objective (NA 1.40, oil immersion).
For transmission electron microscopy (TEM), chitosan-phage nanoparticles were prepared by a dilution 1:5 v/v in ultrapure water.
Afterwards, a 10 μL droplet was deposited on a cooper grid coated with
a carbon layer. The droplet was absorbed by a filter paper after 30 s in
contact with the grid and left to evaporate in air at room temperature.
Uranyl acetate at 2% was employed as positive staining for the noncoated phage. The morphological examination of chitosan-encapsulated
bacteriophage nanoparticle was carried out in a JEOL (JEM 1200 EX II,
Tokyo, Japan) with an accelerating voltage of 100 kW. The images were
recorded with a CCD camera (Orius BioScan Model 792) and software
Gatan digital micrograph at a resolution of at least 2004 × 1335 pixels.

To identify aflatoxin mimotopes, phage-displayed peptide libraries
were selected by affinity using anti-AFB1 specific antibodies as schematically represented in Fig. 1A. Three rounds of selection were performed and the reactivity of the amplified phage pool of each panning
was assessed by ELISA (Fig. 1B). A significant enrichment of phage
affinity was obtained after three rounds of panning, indicated by a 102
pfu mL−1 reduction on phage recovery between first and second
rounds. Otherwise, the reactivity of the phage eluted increased after the
third panning, being eight-fold higher than those in the second panning.
Individual clones were obtained after screening based on the ability
to bind to anti-AFB1 monoclonal antibody (Fig. 1C). Considering 82
clones randomly selected from phage pannings, nine of them were recognized for binding to antibodies against aflatoxin, exhibiting reactivity at least 20-fold higher than WTP. These results indicate that the
selected phage clones reactivity occurs merely between antibodies and

peptides fused to coat protein on phage particles.
3.2. Immunological properties of mimotopes selected from random phagedisplayed peptide libraries
The most reactive clones against anti-AFB1 antibodies were amplified and titrated for assessment of their specificity, comparing the results obtained from WTP and irrelevant phage 3P25. The specificity was
defined by the ability of a clone to be identified only by its cognate
target − anti-AFB1 monoclonal (mAb anti-AFB1) and polyclonal antibodies (pAb anti-AFB1) − (Fig. 2A) among different irrelevant ligands
(Fig. 2B). Any clones showed specificity for BSA or irrelevant murine
IgG. However, some clones − 3P4, 3P5 and 3P16–exhibited recognition
against the mAb7 monoclonal and rabbit polyclonal antibodies, which
indicates that these clones were less specific than other selected clones.
The affinity-selected phages exhibited a concentration-dependence
profile: the reduction on phage concentration from 1011 to
108 pfu mL−1 causes a decreased reactivity towards the anti-AFB1

2.2.3.3. Immunization of mice with phage nanoparticles. The
immunogenicity of chitosan-phages formulations was assessed in
Swiss mice following intranasal immunization. Thirty micrograms of
antigen (1011 phages mL−1 associated or not with 1 mg mL−1 of
chitosan) in 10 μL of PBS were administered in the animal nostrils
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Fig. 2. Evaluation of the selectivity and specificity of the selected clones. (A) Reactivity of specific phage clones (3P4–3P30) and irrelevant phage clone (3P25) at 1012 pfu mL−1 with antiAFB1 antibody demonstrated as mean absorbance (490 nm) ± SD. The specificity of the phage clones was determined as absorbance values from anti-AFB1 monoclonal and polyclonal
antibodies. (B) Reactivity of specific phage clones (3P4–3P30) and irrelevant phage clone (3P25) at 1012 pfu mL−1 with different targets demonstrated as mean absorbance
(490 nm) ± SD. (C) Reactivity of specific phage clones (3P4–3P30) and irrelevant phage clone (3P25) at 1011, 1010, 109, and 108 pfu mL−1 with anti-AFB1 antibody demonstrated as
mean absorbance (490 nm) ± SD.

that they correspond to different specific binding sites of anti-aflatoxin
antibodies (Thirumala‐Devi, Miller, Reddy, Reddy, & Mayo, 2001; Liu
et al., 2012; Wang et al., 2013).
The amino acid sequence was chemically synthesized including a
terminal cysteine residue to be coupled to BSA and ovalbumin (OVA),
via SMCC (Thermo Scientific, Waltham, USA). To assess the protein
profile of each coupled system, the proteins were submitted to electrophoresis and the gel was stained with silver. The difference in
electrophoretic mobility indicates coupling between the peptide and
the carrier proteins, as visualized by the molecular mass increment
from BSA (native protein) to peptide-BSA (coupled protein) (Fig. 1,
Supplementary data). Based on this result, BSA carrier peptide was used
as immunogen in mice.

monoclonal antibody, while any interference was observed with the
irrelevant phage 3P25 or WTP (Fig. 2C). These results also indicate that
the phage-displayed peptides represented the binding site of the
aforementioned antibody.
3.3. Bioinformatics analysis and characterization of synthetic peptide
immunogen
The respective phage clones had their sequences identified and an
alignment showed that identical consensus motif were detected among
them. These peptides were selected from the X15 library, which expressed linear peptides with 15 residues. In addition, the sequence 3P25
identified as non-specific binder to anti-AFB1 antibody presented a
completely different sequence and was obtained from the 17-mer library (C8 × C8).
The mimotope peptide sequence QTDLDYLHPLINSWN, with a
molar mass of 1825 Da and a theoretical isoelectric point of 3.91 was
deduced using the Expasy server. This sequence exhibits hydrophobic
uncharged residues, such as lysine, proline and tryptophan, which
contributed to increase the hydrophobicity − up to 40% − of the sequence and displayed partial water solubility. The comparison of the
selected mimotope sequence with peptide sequence databases did not

reveal any significant similarity with amino acid sequences of phage
clones previously selected with anti-aflatoxin antibodies, suggesting

3.4. Immunogenicity of mimetic aflatoxin immunogens
The immunological in vitro results of selected mimotopes showed
that selected phage clones were able to mimic the AFB1 epitope recognition by the anti-AFB1 antibody. Next, we addressed their immunogenicity potential, i.e. their ability to induce antibodies that recognize native epitopes. Groups of mice were injected with synthetic
peptide conjugated with BSA, AFB1-BSA and phage 3P30, an irrelevant
phage 3P25, randomly chosen from the very-low reactivity group, and
WTP were injected as controls (Fig. 3). The selected phage 3P30 was
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C.Y.T. de Andrade et al.

Fig. 3. Immunogenicity of selected phage clones and synthetic peptide.
Indirect ELISA antibody titer in sera from mice immunized with peptideBSA, AFB1-BSA, specific phage clone 3P30, irrelevant phage clone 3P25 or
wild type phage (WTP). Mean values and SD of the reciprocal titer of each
treatment group are indicated. The one way Anova followed by a Tuckey’s
test were used, and * represent p < 0.05 (column in blue) and a, b and c
are different (column in red) (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

Fig. 4. Confocal fluorescence microscopy images of phage 3P30 coating with chitosan, after centrifugation. FITC-labeled chitosan (green), Alexa fluor 633-conjugated anti-IgG murine
labeled phage (red) and co-localization of chitosan and phages (yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)

anti-peptide antibody titer was at least 2-fold higher compared with the
groups immunized with AFB1, 3P30, 3P25 or WTP. The group treated

with AFB1-BSA produced higher contents of anti-AFB1 antibodies than
groups immunized with 3P30, 3P25 or WTP. Interestingly, the mice
immunized with peptide-BSA were also induced to produce such anti-

randomly chosen among specific phage clones (3P8, 3P13, 3P16, 3P19,
3P20, 3P23 and 3P30), to represent the phage bearing aflatoxin mimotope.
The ELISA results revealed the presence of anti-peptide antibodies
in all groups analyzed. For the group immunized with the peptide, the
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C.Y.T. de Andrade et al.

phages displayed with higher electron-density contrast. Therefore, the
confocal and electronic images confirmed that the polymer was able to
individually recover the phages at the nanoscale (TEM images) and
simultaneously provide a micrometer-size platform of chitosan (confocal images), which configures an attractive delivery system to be intranasaly administered.
After entrapping the phage into chitosan, the apparent hydrodynamic radius (Rhapp) increased from ∼130 nm for bare phage to
∼345 nm and 445 nm at pH 4.6 and 7.0, respectively (Table 1). Additionally, the presence of a positively charged layer caused the ζ-potential to the phage to vary from −30 mV to +40 mV at pH 4.6 and
from −80 mV to +20 mV at pH 7.0. Altogether, the results indicate an
effective encapsulation process of the phage, which completely altered
the phage’s surface properties.
The values of pH 4.6 and 7.0 were measured for Rh and zeta potential (Table 1), since initially the phage was dispersed at acetate
buffer pH 4.6 to induce a complex coacervation. Only after this procedure the pH was raised to pH 7.0, promoting the complex precipitation of a chitosan layer on phages. Increasing the pH from 4.6 to
7.0 the zeta potential of phages reduced, due to ionization of proteins at
phage surface. For chitosan and the phage-coated with chitosan the
potential also diminish due to the reduction of amine group ionization
effect (pKa ∼6.5). In parallel the apparent Rh reduced for the phage,

and increased for chitosan and phage-coated chitosan, compatible with
the complex coacervation at pH 4.6 and complex precipitation at pH
7.0.

AFB1 antibodies, though in lower amounts than the AFB1-BSA group.
This result highlights the ability of the mimetic peptide to induce a
specific humoral immune response with production of anti-AFB1 antibodies without exposure to the toxin.
3.5. Physicochemical properties and association efficiencies of chitosanbacteriophage nanoparticles
The selected phage 3P30 was coated with chitosan polymer, with
the intent to enhance the immune response based on the synergistic
effect with the biopolymer, associated with an increasing residence in
the intranasal mucosa and phage mucosa absorption. After the chitosanprecipitation process, the phages that were encapsulated into the
polymer’s nanoparticles precipitated once centrifuged, demonstrating
distinct physical-chemical properties endowed by chitosan complexation with phages.
The efficiency of phage entrapment was determined by the amount
of phages remaining in the supernatant after centrifugation. From noncoated phages 100% of phages remained in the supernatant, and in the
presence of chitosan there were almost any signal of the phage
(< 5 pfu mL−1), which are in the limit of detection of our technique to
quantify phage. The LC was 1,78 × 1014 pfu/g of chitosan particles.
Several controls experiments, without the presence of chitosan and
in the same experimental conditions employed for nanoparticle synthesis, almost all phages content (> 1011 pfu mL−1) remain in the supernatant after centrifugation. This result indicates that the procedure
of chitosan nanoparticle preparation did not negatively affect the antigen, maintaining its viability along the process.
To analyze the macromolecular organization of the nanoparticles
embedded system, we employed a FITC-labeled chitosan and a secondary antibody coupled to Alexa fluor 633 to label the phage 3P30.
The confocal microscopy images displayed the presence of chitosan
agglutinates in green that were loaded with the phage in red (Fig. 4).
The phage co-localization inside chitosan was random as verified by zstacking merge image, in yellow (Fig. 4). This clearly demonstrated the
phage 3P30 coating with chitosan. The control of FITC-chitosan particles were also performed, and presented in the supplementary information (Fig. 6).
TEM images of diluted samples stained with uranyl acetate revealed
a well-defined phage structure with positive contrast only at the capsid

shell (Fig. 5A). The phage coated with chitosan, however, displayed
structures with higher electron-density and similar aspect ratio, with an
average length of 450 nm, as shown by the uncoated phage (Fig. 5B and
C). The polymer favored the contour delineation and contrast of the
phages coated with chitosan, compared to uncoated phage. These results indicate that chitosan has homogenously recovered the entire
phage surface. Therefore, individual chitosan-shelled phages were
successfully visualized by TEM and the filamentous-coated phages are
probably randomly distributed along the matrix bed.
The more spherical aggregates could be associated to imperfections
of the foil of carbon. The filamentous phages are much more evident in
the image, because of the contrast obtained with uranyl acetate. In
Fig. 5B, chitosan favored the contour delineation and contrast of the

3.6. Mice immunization with chitosan-phage nanoparticles
All mice immunized with three doses of the chitosan-encapsulated
bacteriophages did not show any sign or symptoms of adverse effects in
Mice, mainly based in the behavior of the animals during the experiments. To investigate the suitability of the antigen coated with chitosan, we compared the serum responses of mice after intranasal administration of antigen alone, antigen coated with chitosan or soluble
antigen co-administered with chitosan solution with a 2 h interval.
After intranasal immunization protocol, phage 3P30 coated with chitosan exhibited a 2.5-fold higher immunogenicity than the free antigen
(Fig. 6A). As shown in Fig. 6B, the anti-peptide IgG levels elicited by
chitosan-phage 3P30 nanoparticles were higher than those corresponding to the phage solution.
In pulse-chase study, the mice that received chitosan solution before
the bacteriophage solution developed weak IgG titers, indicating that
chitosan is less effective when administered 2 h prior intranasal administration of phage. Therefore, the adjuvant effect is mainly based on
the phage improved delivery by chitosan than due to immune stimulation by chitosan by it self. Accordingly, bare chitosan showed any
antibody titers after intranasal administration.
The results of IgA anti-peptide and anti-AFB1 antibodies in
bronchoalveolar lavages are shown in Fig. 6C. As the humoral response,
the IgA levels produced by chitosan-coated bacteriophage were higher
than those corresponding to the free antigen likewise indicated a sitespecific antibody induction. Altogether, the results corroborate that

Fig. 5. Transmission electron microscopy (TEM) images of pure 3P30 phage at 40.000× of magnification stained with 2% uranyl acetate (A) and after coating into chitosan nanoparticles
with 8000× (B) and several regions with 40.000× of magnification, respectively (C).

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C.Y.T. de Andrade et al.

Table 1
Apparent hydrodynamic radius and ζ-potential of phages and chitosan-coated phages at
different pH values. All results represent the average of 5 independent measurements.
Sample

Phage
Chitosan
Chitosan-Phage

ζ-Potential (mV)

Rhapp (nm)*
pH 4.6

pH 7.0

pH 4.6

pH 7.0

161 ± 3
89 ± 15
345 ± 26

116 ± 2
250 ± 80
444 ± 150

−33 ± 6
+35 ± 6
+40 ± 5

−80 ± 10
−10 ± 2
+20 ± 3

4. Discussion
The aims of this work were to identify possible peptides mimetic of
AFB1, investigate their properties in comparison with the original
epitope and employ them as immunogen for mucosal vaccine against
aflatoxicosis. Particularly, the ability of phage-displayed peptides to act
as antigenic mimotopes was demonstrated in many reports (Ramada
et al., 2013; Alban, Moura, Minozzo, Mira, & Socool, 2013; Fogaỗa
et al., 2014). Based on genetic engineering of bacteriophages, as well as
repeated rounds of antigen-guided selection and phage propagation,
this approach offers an in vitro selection from any specific target (Scott
& Smith, 1990). These characteristics make the phage display technology a powerful and cost-effective method for identifying peptides,
which are able to bind to the target with high affinity and specificity
(Huang et al., 2011).

Initially, 4 distinct libraries were screened and phage selection was
performed by solid-phase with decreasing amount of the target, additional washings and elution by sonication (Lunder et al., 2008). Although these libraries presented a variation of 8–17 amino acid residues, only the one that presented 15-mer peptides generated the best
mimotope binding efficiency with anti-AFB1 monoclonal antibodies.
Based on the immunoassay results, high-quality mimotopes with the
ability to mimic the basic functions of the epitope, such as recognition
and antigenicity were obtained. This effect was not verified towards the
irrelevant phage 3P25 or wild type phage, demonstrating that the selected peptides mimicked in vitro immunological characteristics correspondent to AFB1 epitope region.
According to bioinformatic analysis it was possible to determine
that the amino acid sequence of selected mimotopes is different from
previous studies using other anti-aflatoxin monoclonal antibodies
(Thirumala‐Devi et al., 2001). In particular, the binding efficiently of
our mimotope is up to 8-fold higher compared to recent reports of Liu
et al. (2012) and Wang et al. (2013), a reflect from the panning
strategy. Such performance may be a result from the selection of mimotopes with lower dissociation constants with antibodies promoted by
the sonication process along the panning phase as previously discussed
by Lunder et al. (2008). However, all peptide sequences obtained so far
exhibit hydrophobic domains correspondent to aromatic amino acid
residues, which may reflect a degree of molecular mimicry by the ring
structures in the aflatoxin molecules.
Although some studies produced aflatoxin mimetic peptides, any of
them has translated this technology to the in vivo immunization modeling. This strategy of peptides obtained by phage display to induce
protection against toxins was confirmed by previous studies (de Moura
et al., 2011, Sagona et al., 2016), so we sought to explore the potential
of synthetic peptide and mimotope to induce an immune response
against aflatoxicosis. To our knowledge, this is the first report that
employs aflatoxin mimetic peptides obtained by phage display as epitope-based vaccines.
The initial in vivo experiments demonstrated that both the peptide
and the phage 3P30 were able to induce the production of anti-AFB1
antibodies in mice, thus, indicating the proof of concept for aflatoxin
mimicry. This strategy reflects the development and refinement of

phage display technology, wherein phage-displayed peptide ligands of
monoclonal antibody were also generated for immunization purposes,

Fig. 6. Immunogenicity of selected phage clones encapsulated by chitosan nanoparticles.
Indirect ELISA antibody titer in sera and bronchoalveolar lavages from mice immunized
with: chitosan-phage 3P30 nanoparticle; phage solution 3P30; chitosan solution 2 h before phage solution 3P30; chitosan-WTP nanoparticle; WTP; or chitosan solution.
Reciprocal titers of 3P30, WTP and 3P25 (A), specific IgG anti-peptide and anti-AFB1 (B),
and specific IgA anti-peptide and anti-AFB1 (C). Mean values and SD of the reciprocal
titer of each treatment group are indicated. The one way Anova followed by a Tuckey’s
test were used, and * represent p < 0.05.

chitosan-encapsulated phages provide a more specific mucosal immune
response compared to non-coated phages. These results clearly demonstrated that chitosan-coated phages 3P30 are much more effective
to induce immunization than bare phages 3P30, or chitosan itself.

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C.Y.T. de Andrade et al.

charged materials, such as cell surface of phages or mucosa mucus,
promoting the coating (mucus contain significant proportion of sialic
acid). At physiological pH, sialic acid carries a negative charge, and as
consequence, mucin and chitosan can demonstrate strong electrostatic
interactions. The complexes between chitosan and mucin are highlighted by electrostatic interactions crucial for the mucoadhesive mechanism (Silva, Nobre, Pavinatto, & Oliveira, 2012).
Menchicchi et al. (2014) described that the interaction between
chitosan and mucin contract the gel network on the mucosa, and thus
creates large pores throughout the gel mesh. In this case, the antigen

adsorption could be enhanced, increasing the immunological response.
Based on this explanation, the synergistic effect of chitosan could be
associated to increase in the nasal residence and absorption, that should
increase phage-chitosan uptake by the M-cells, responsible for the uptake of virus, toxin and microparticles < 10 μm. After, the pathogens
could be transported to NALT (Nasal Associated Lymphoid Tissue), just
below the epithelium surfaces that contain B-Cell areas, T-Cell areas,
macrophages and dendritic cells (Kuper et al., 1992; Cesta, 2006), inducing the specific immune responses.

with the goal of eliciting anti-peptide antibodies that also recognize the
native antigen (Henry, Arbabi-Ghahroudi, & Scott, 2015). In particular,
the use of phage-conjugate peptides as immunogens are advantageous
compared to free peptides by assuming a favorable conformation to act
as binder of antibodies, exposing in a more efficient manner the recognizable regions when compared to the free synthetic peptide (Henry,
Murira, van Houten, & Scott, 2011).
Nevertheless, because of the small molecular weight and low immunogenicity, epitope-based vaccines usually require the use of adjuvants to increase antigen-specific immune responses (Henry et al.,
2015). Such adjuvants (e.g. proteins, liposomes or nanoparticles) are
ubiquitous to allow phage or peptides to trespass biological barriers
(e.g. mucous layers), increase residence time in the bloodstream and
enhance specific host-recognition (Sun & Xia, 2016). For this purpose,
chitosan a natural, biocompatible and biodegradable polymer that has
been used to deliver antigens across different mucosal surfaces (Yoo
et al., 2010). In fact, many studies highlight the chitosan ability to
strongly adhere to the epithelium and facilitate the opening of intercellular tight junctions, enhancing the transport of antigens through the
nasal airways (Jiang et al., 2004).
To increase the specific immune response efficiency and improve
the phage immunogenicity, we proposed the entrapment of the phage
3P30 into a chitosan-shell for nasal delivery. The coating with chitosan
has completely altered the properties of the phage, causing its sedimentation upon centrifugation, and clearly demonstrating 100% of
encapsulation efficiency. This effect might be correlated with the acquired mass gained by the phage as it was entrapped into the macromolecular mesh of the polymer. Thus, particles that presented a reduced time-of-flight compared to nanostructures − such as the bare
phage − were produced and demonstrate a greater potential to carry

the phages throughout the airways. In addition, because of the negative
charge of the phage at pH 4.6 (–33 mV), it offered an optimal template
to interact electrostatically with chitosan, positively charged at this
condition (+35 mV), which was able to coat individual phages − that
ultimately exhibited similar charge as chitosan (+40 mV) at pH 4.6.
The apparent size increment to ∼345 nm was a reflection of the phage’s
polymer coating.
As the polymer was able to individually recover the phages at the
nanoscale and simultaneously provide a micrometer-size platform of
chitosan, it configures as an attractive delivery system to be administered intranasally. Based on the adherence properties as well as the
higher density and weight proportioned by macromolecular organization assumed by chitosan after the precipitation process, an increased
absorption, and a more efficient exposition of the peptide to the immune system was expected for phages. Indeed, the antigen-loaded
chitosan nanoparticles fully retained the immunogenicity of the original immunogen. Since nanometric objects are characterized by a low
inertia and, consequently, rapid nasal exhalation, the encapsulation
into chitosan nanoparticles embedded into higher mass aggregates was
helpful to provide higher phage contents at the bloodstream, probably
because of the polymers ability in anchoring to epithelium and slowly
dissociate, releasing the phages (Tsapis, Bennett, Jackson, Weitz, &
Edwardz, 2002).
Chitosan are suggested to be an excellent vehicle for nasal mucosa
administration, increasing the phage nasal residence. According to Van
der Lubben, Verhoef, Borchard, & Junginger (2001) the nasally administered vaccines have to be transported over very small distances,
remaining only about 15 min in the nasal cavity due to chitosan
coating, reducing the exposure to low pH values and degradation enzymes. Bacon et al. (2000) reported that chitosan is able to enhance
both the mucosal and systemic immune responses against influenza
virus vaccines, and only mice which received chitosan vaccines formulation intranasally could develop high immunoglobulin titer in the
nasal washings. The results of 3P30 phage coated with chitosan pointed
in the same directions as presented above.
Due to cationic nature, chitosan strongly binds to negatively

5. Conclusion
In conclusion, the experiments performed in this study using the
aflatoxin mimotopes showed that high affinity mimotopes represented
individual binding sites of the antibody. After immunization with
phage, an improved specific in vivo immune response was provided,
which demonstrated the value of phage display technology to engineer
phage-conjugated peptides as immunogen for carcinogenic haptens
such as aflatoxins. The chitosan acted as important adjuvant in nasal
formulations, without immunogenic activity, but increasing the immune response and the residence of aflatoxin mimotopes in the nasal
mucosa. Chitosan appeared to be an excellent vehicle for phages vaccines in vivo.
Acknowledgements
We acknowledge Electron Microscopy Center and Confocal
Laboratory of Federal University of Paraná for the technical support.
Statement of funding: The present research was supported by the
Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq), n° 314441/2014-0. This work was also supported by funds
granted by CAPES (Coordenaỗóo de Aperfeiỗoamento de Pessoal de
Nível Superior − Ministry of Education, Brazil).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
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