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.
Trang 1Contents lists available atScienceDirect
Carbohydrate Polymers journal homepage:www.elsevier.com/locate/carbpol
Physicochemical and immunological characterization of chitosan-coated
bacteriophage nanoparticles for in vivo mycotoxin modeling
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 Limq, Basic Patology Department, Federal University of Paraná, 81530-900 Curitiba, PR, Brazil
b Biopol, Chemistry Department, Federal University of Paraná, 81531-980 Curitiba, PR, Brazil
A R T I C L E I N F O
Keywords:
Phage display
Mimotope
Aflatoxin B1
Peptide carrier
Mucosal vaccine
Chitosan
A B S T R A C T
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 anti-AFB1 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 de-livery 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 epi-tope-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
in-toxication, 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
afla-toxins 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
chroni-cally exposed to high amounts of aflatoxins and the intake of such
toxins over a long period of time, even at low concentrations,
sig-nificantly increases the risk of hepatocellular carcinoma and
extra-hepatic tumors (Gnonlonfin et al., 2013) AFB1 has a wide range of
biological activities, including genotoxicity, teratogenicity, hepato-toxicity, nephrotoxicity and immunosuppression (Wild et al., 2015)
As animals ingest aflatoxin-contaminated grains, important para-meters 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., 2011andGiovati et al., 2014) The AFB1-derived vaccines, however, have been reported to produce a limited im-munogenicity 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
https://doi.org/10.1016/j.carbpol.2017.12.063
Received 29 September 2017; Received in revised form 6 December 2017; Accepted 22 December 2017
⁎ Corresponding authors.
E-mail addresses: rilton@ufpr.br , rilton@quimica.ufpr.br (R.A de Freitas), lmalvarenga@ufpr.br (L.M Alvarenga).
Available online 28 December 2017
0144-8617/ © 2017 Elsevier Ltd All rights reserved.
T
Trang 2relies on the replacement of the toxin by mimotopes, i.e
peptide-dis-played 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 offilamentous 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
pep-tide 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
re-garding its immunogenic properties The phage-displayed mimotope
and its synthetic peptide were able to induce humoral response in
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, Remuñán López, & Grenha, 2012) extensively used as phar-maceutical 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 N-acetyl-D-glucosamine units bonded viaβ-(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 chitosan-shelled phages provided a more efficient specific immune response compared to non-coated phages In this context, antigen identified as a
Fig 1 Selection of phage-displayed peptides recognized by AFB1 antibodies (A) Schematic representation of panning procedure Phage display libraries were screened against anti-AFB1 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.
Trang 3mimotope of a non-proteinaceous molecule may be considered a
pro-spect 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
al-bumin (BSA); BSA conjugated with AFB1; Freund’s complete and
in-complete adjuvant; O-phenylenediamine dihydrochloride (OPD); acetic
acid; sodium acetate; NaOH; isothiocianate offluoresceine (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
an-tibody anti-IgG murine (Thermo Scientific, Waltham, USA); coopergrid
coated with a carbon layer (Pelco, Clovis, USA); Uranyl acetate
(Poly-sciences, Warrington, USA) Chitosan was obtained from Purifarma
(São Paulo, Brazil)
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 Phage display
The panning procedure (Fig 1A) was performed as previously
de-scribed byScott & Smith (1990)andLunder, 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 20μg mL−1tetracycline The supernatants containing phages were
obtained by centrifugation (1.6 × 103g, 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
O-phenylenediamine 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)
2.2.2.2 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 byCapelli-Peixoto
et al (2011), in detail in the Supplementary data
2.2.2.3 Immunization of mice with phage and synthetic peptide Immunization of mice with phages was performed as described byGalfrè et al (1996) Briefly, phage clone 3P30 (1 × 1011 particles) in 100μL 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, Sigma-Aldrich, USA) or synthetic peptide (25μg dissolved in 50 mmol L−1 phosphate buffer saline, PBS, 150 mmol L−1NaCl, pH 7.4) The mice belonging to the control groups were injected with WTP or with the irrelevant phage 3P25 Allfive 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 byfinal 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
2.2.2.4 Indirect ELISA for determination of anti-peptide and anti-AFB1 antibodies Microtitration plates were coated at 4 °C for 16 h with
10μg mL−1of 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 anti-IgG 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
2.2.3 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 × 105g 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 by1H 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 × 1013pfu mL−1 and afinal solution was obtained after dilution in 10 mL ultrapure water to a concentration of
1 × 1011pfu mL−1 The phage coating with chitosan was performed using the coa-cervation/precipitation processes Thefirst 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 offinal solution of phages into 10 mL of chitosan in 0.01 mol L−1
Trang 4acetic acid/sodium acetate buffer, 1 mg mL−1, pH 4.6, under
con-tinuous mild stirring After complete mixture the solution was
main-tained in agitation for at least 1 h The solution was then neutralized to
pH 7.0 with a 0.2 mol L−1NaOH solution, under continuous agitation
The chitosan coated-bacteriophage were centrifuged at 104g, 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
= ⎛
⎝
⎠
EE total phages free phages
total phages
(1)
= ⎛
⎝
⎠
LC total phages free phages
The total phages was the initial phages added (1,78 × 1012pfu),
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
chit-osan 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
2.2.3.2 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
na-noparticles were prepared by a dilution 1:5 v/v in ultrapure water
Afterwards, a 10μL droplet was deposited on a cooper grid coated with
a carbon layer The droplet was absorbed by afilter 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
non-coated 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
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 10μL of PBS were administered in the animal nostrils
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 1011pfu mL−1 Group 2: Free phage 3P30 at 1011pfu 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 1011pfu 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 1011pfu mL−1 Group 5: Free phage WTP at
1011pfu 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 post-administration and serum samples were maintained at −20 °C prior ELISA analysis as described above Microtitration plates were coated with 10μg mL−1 of BSA, AFB1-BSA and peptide-BSA as with
1011pfu mL−1 of the phage 3P30, WTP or irrelevant phage 3P25 Bronchoalveolar lavages were collected 38 days after the last adminis-tration, using a modified procedure described byVila et al (2003), in detail in the Supplementary data
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
To identify aflatoxin mimotopes, phage-displayed peptide libraries were selected by affinity using anti-AFB1 specific antibodies as sche-matically represented inFig 1A Three rounds of selection were per-formed 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 cognized for binding to antibodies against aflatoxin, exhibiting re-activity 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 phage-displayed peptide libraries
The most reactive clones against anti-AFB1 antibodies were ampli-fied and titrated for assessment of their specificity, comparing the re-sults 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− AFB1 monoclonal (mAb AFB1) and polyclonal anti-bodies (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
108pfu mL−1 causes a decreased reactivity towards the anti-AFB1
Trang 5monoclonal 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
ex-pressed 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
li-brary (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
se-quence 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
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 elec-trophoresis 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
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 re-cognition by the anti-AFB1 antibody Next, we addressed their im-munogenicity potential, i.e their ability to induce antibodies that re-cognize 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
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 10 12 pfu mL−1with anti-AFB1 antibody demonstrated as mean absorbance (490 nm) ± SD The specificity of the phage clones was determined as absorbance values from anti-anti-AFB1 monoclonal and polyclonal antibodies (B) Reactivity of specific phage clones (3P4–3P30) and irrelevant phage clone (3P25) at 10 12 pfu mL−1with different targets demonstrated as mean absorbance (490 nm) ± SD (C) Reactivity of specific phage clones (3P4–3P30) and irrelevant phage clone (3P25) at 10 11 , 10 10 , 10 9 , and 10 8 pfu mL−1with anti-AFB1 antibody demonstrated as mean absorbance (490 nm) ± SD.
Trang 6randomly chosen among specific phage clones (3P8, 3P13, 3P16, 3P19,
3P20, 3P23 and 3P30), to represent the phage bearing aflatoxin
mi-motope
The ELISA results revealed the presence of anti-peptide antibodies
in all groups analyzed For the group immunized with the peptide, the
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-Fig 3 Immunogenicity of selected phage clones and synthetic peptide Indirect ELISA antibody titer in sera from mice immunized with peptide-BSA, AFB1-peptide-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 re-ferences 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.)
Trang 7AFB1 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 AFB1
anti-bodies without exposure to the toxin
3.5 Physicochemical properties and association efficiencies of
chitosan-bacteriophage 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
chitosan-precipitation process, the phages that were encapsulated into the
polymer’s nanoparticles precipitated once centrifuged, demonstrating
distinct physical-chemical properties endowed by chitosan
complexa-tion with phages
The efficiency of phage entrapment was determined by the amount
of phages remaining in the supernatant after centrifugation From
non-coated 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 × 1014pfu/g of chitosan particles
Several controls experiments, without the presence of chitosan and
in the same experimental conditions employed for nanoparticle
synth-esis, almost all phages content (> 1011pfu mL−1) remain in the
su-pernatant after centrifugation This result indicates that the procedure
of chitosan nanoparticle preparation did not negatively affect the
an-tigen, maintaining its viability along the process
To analyze the macromolecular organization of the nanoparticles
embedded system, we employed a FITC-labeled chitosan and a
sec-ondary antibody coupled to Alexafluor 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
z-stacking merge image, in yellow (Fig 4) This clearly demonstrated the
phage 3P30 coating with chitosan The control of FITC-chitosan
parti-cles were also performed, and presented in the supplementary
in-formation (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
re-sults indicate that chitosan has homogenously recovered the entire
phage surface Therefore, individual chitosan-shelled phages were
successfully visualized by TEM and thefilamentous-coated phages are
probably randomly distributed along the matrix bed
The more spherical aggregates could be associated to imperfections
of the foil of carbon Thefilamentous 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
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 (con-focal images), which configures an attractive delivery system to be in-tranasaly administered
After entrapping the phage into chitosan, the apparent hydro-dynamic 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) Ad-ditionally, the presence of a positively charged layer caused the ζ-po-tential 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 Rhand zeta po-tential (Table 1), since initially the phage was dispersed at acetate buffer pH 4.6 to induce a complex coacervation Only after this pro-cedure the pH was raised to pH 7.0, promoting the complex pre-cipitation 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 Rhreduced 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
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 experi-ments To investigate the suitability of the antigen coated with chit-osan, we compared the serum responses of mice after intranasal ad-ministration 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 chit-osan exhibited a 2.5-fold higher immunogenicity than the free antigen (Fig 6A) As shown inFig 6B, the anti-peptide IgG levels elicited by chitosan-phage 3P30 nanoparticles were higher than those corre-sponding 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 ad-ministration of phage Therefore, the adjuvant effect is mainly based on the phage improved delivery by chitosan than due to immune stimu-lation 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 inFig 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 site-specific 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).
Trang 8chitosan-encapsulated phages provide a more specific mucosal immune
response compared to non-coated phages These results clearly
de-monstrated that chitosan-coated phages 3P30 are much more effective
to induce immunization than bare phages 3P30, or chitosan itself
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; Fogaça
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 tech-nology 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, addi-tional washings and elution by sonication (Lunder et al., 2008) Al-though these libraries presented a variation of 8–17 amino acid re-sidues, 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 se-lected peptides mimicked in vitro immunological characteristics corre-spondent 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 ofLiu
et al (2012) and Wang et al (2013), a reflect from the panning strategy Such performance may be a result from the selection of mi-motopes with lower dissociation constants with antibodies promoted by the sonication process along the panning phase as previously discussed
byLunder 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 mod-eling 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 epi-tope-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
be-fore 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.
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 R happ (nm)* ζ-Potential (mV)
pH 4.6 pH 7.0 pH 4.6 pH 7.0 Phage 161 ± 3 116 ± 2 −33 ± 6 −80 ± 10 Chitosan 89 ± 15 250 ± 80 +35 ± 6 −10 ± 2 Chitosan-Phage 345 ± 26 444 ± 150 +40 ± 5 +20 ± 3
Trang 9with 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
re-cognizable regions when compared to the free synthetic peptide (Henry,
Murira, van Houten, & Scott, 2011)
Nevertheless, because of the small molecular weight and low
im-munogenicity, epitope-based vaccines usually require the use of
ad-juvants 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
inter-cellular 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
sedi-mentation upon centrifugation, and clearly demonstrating 100% of
encapsulation efficiency This effect might be correlated with the
ac-quired mass gained by the phage as it was entrapped into the
macro-molecular mesh of the polymer Thus, particles that presented a
re-duced 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
adminis-tered intranasally Based on the adherence properties as well as the
higher density and weight proportioned by macromolecular
organiza-tion assumed by chitosan after the precipitaorganiza-tion process, an increased
absorption, and a more efficient exposition of the peptide to the
im-mune system was expected for phages Indeed, the antigen-loaded
chitosan nanoparticles fully retained the immunogenicity of the
ori-ginal 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 toVan
der Lubben, Verhoef, Borchard, & Junginger (2001) the nasally
ad-ministered 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
en-zymes.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
for-mulation 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
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 high-lighted by electrostatic interactions crucial for the mucoadhesive me-chanism (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 up-take of virus, toxin and microparticles < 10μm 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), in-ducing the specific immune responses
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 im-mune response and the residence of aflatoxin mimotopes in the nasal mucosa Chitosan appeared to be an excellent vehicle for phages vac-cines 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 (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior− Ministry of Education, Brazil)
Appendix A Supplementary data
Supplementary data associated with this article can be found, in the online version, athttps://doi.org/10.1016/j.carbpol.2017.12.063
References
Alban, S M., de Moura, J F., Minozzo, J C., Mira, M T., & Soccol, V T (2013) Identification of mimotopes of Mycobacterium leprae as potential diagnostic re-agents BMC Infectious Diseases, 13, 42
Alvarenga, L M., Martins, M S., Moura, J F., Kalapothakis, E., Oliveira, J C., Mangili, O C., et al (2003) Production of monoclonal antibodies capable of neutralizing der-monecrotic activity of Loxosceles intermedia spider venom and their use in a specific immunometric assay Toxicon, 42, 725–731
Bacon, A., Makin, J., Sizer, P J., Jabbal-Gill, I., Hinchcliffe, M., Illum, L., et al (2000) Carbohydrate biopolymers enhance antibody responses to mucosally delivered vac-cine antigens Infection and Immunity, 68(10), 5764–5770
Baek, M., Lee, J A., & Choi, S J (2012) Toxicological effects of a cationic clay, mon-tmorillonite in vitro and in vivo Molecular & Cellular Toxicology, 8, 95–101
Bonnycastle, L L C., Mehroke, J S., Rashed, M., Gong, X., & Scott, J K (1996) Probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed
by filamentous phage Journal of Molecular Biology, 258, 747–762
Capelli-Peixoto, J., Chávez-Olórtegui, C., Chaves-Moreira, D., Minozzo, J C., Gabardo, J., Teixeira, K N., et al (2011) Evaluation of the protective potential of a Taenia solium cysticercus mimotope on murine cysticercosis Vaccine, 29, 9473–9479
Casettari, L., & Illum, L (2014) Chitosan in nasal delivery systems for therapeutic drugs.
Trang 10Journal of Controlled Release, 190, 189–200
Cesta, M F (2006) Normal structure, function, and histology of mucosa-associated
lymphoid tissue Toxicologic Pathology, 34(5), 599–608
Chang, S E., & Chien, Y W (1984) Intranasal drug administration for systemic
medi-cation Pharmaceutics International, 5, 287–288
de Moura, J., Felicori, L., Moreau, V., Guimarães, G., Dias-Lopes, C., Molina, L., et al.
(2011) Protection against the toxic effects of Loxosceles intermedia spider venom
elicited by mimotope peptides Vaccine, 29, 7992–8001
Fogaça, R L., Capelli-Peixoto, J., Yamanaka, I B., de Almeida, R P M., Muzzi, J C D.,
Borges, M., et al (2014) Phage-displayed peptides as capture antigens in an
in-novative assay for Taenia saginata-infected cattle Applied Microbiology &
Biotechnology, 98, 8887–8894
Galán, A., Comor, L., Horvatić, A., Kuleš, J., Guillemin, N., Mrljak, V., et al (2016).
Library-based display technologies: where do we stand? Molecular BioSystems, 12,
2342–2358
Galfrè, G., Monaci, P., Nicosia, A., Luzzago, A., Felici, F., & Cortese, R (1996).
Immunization with phage-displayed mimotopes Methods in Enzymology, 267,
109–115
Gamage, L N A., Ellis, J., & Hayes, S (2009) Immunogenicity of bacteriophage lambda
particles displaying porcine Circovirus 2 (PCV2) capsid protein epitopes Vaccine, 27,
6595–6604
Gibb, H., Devleesschauwer, B., Bolger, P M., Wu, F., Ezendam, J., Cliff, J., et al (2015).
World Health Organization estimates of the global and regional disease burden of
four foodborne chemical toxins, 2010: A data synthesis F1000Research, 4
Giovati, L., Gallo, A., Masoero, F., Cerioli, C., Ciociola, T., Conti, S., et al (2014).
Vaccination of heifers with anaflatoxin improves the reduction of aflatoxin B1 carry
over in milk of lactating dairy cows Public Library Of Science, 9, e94440
Gnonlonfin, G J B., Hell, K., Adjovi, Y., Fandohan, P., Koudande, D O., Mensah, G A.,
et al (2013) A review on aflatoxin contamination and its implications in the
de-veloping world: A Sub-Saharan African perspective Critical Reviews in Food Science
and Nutrition, 53, 349–365
Henry, K A., Murira, A., van Houten, N E., & Scott, J K (2011) Developing strategies to
enhance and focus humoral immune responses using filamentous phage as a model
antigen Bioengineered Bugs, 2, 275–283
Henry, K A., Arbabi-Ghahroudi, M., & Scott, J K (2015) Beyond phage display:
non-traditional applications of the filamentous bacteriophage as a vaccine carrier,
ther-apeutic biologic, and bioconjugation scaffold Frontiers in Microbiology, 6, 755
Huang, J., Ru, B., & Dai, P (2011) Bioinformatics resources and tools for phage display.
Molecules, 16, 694–709
Illum, L., Jabbal-Gill, I., Hinchcliffe, M., Fisher, A N., & Davis, S S (2001) Chitosan as a
novel delivery system for vacines Advanced Drug Delivery Reviews, 51(1–3), 81–96
Illum, L (1998) Chitosan and its use as a pharmaceutical excipient Pharmaceutical
Research, 15, 1326–1331
Jayakumar, R., Moenon, D., Manzoor, K., Nair, S V., & Tamura, H (2010) Biomedical
applications of chitin and chitosan based nanomaterials −A short Review.
Carbohydrate Polymers, 82, 227–232
Jiang, H L., Park, H L., Shin, H L., Kang, S G., Yoo, S G., Kim, S G., et al (2004) In
vitro study of the immune stimulating activity of an athrophic rhinitis vaccine
as-sociated to chitosan microspheres European Journal of Pharmaceutics and
Biopharmaceutics, 58, 471–476
Kuper, C F., Koornstra, P J., Hameleers, D M., Biewenga, J., Spit, B J., Duijvestijn, A.
M., et al (1992) The role of nasopharyngeal lymphoid tissue Immunology Today,
13(6), 219–224
Liu, Y., & Wu, F (2010) Global burden of aflatoxin-induced hepatocellular carcinoma: A
risk assessment Environmental Health Perspectives, 118, 818
Liu, R., Xu, L., Qiu, X., Chen, X., Deng, S., Lai, W., et al (2012) An immunoassay for
determining aflatoxin B1 using a recombinant phage as a nontoxic coating conjugate.
Journal of Food Safety, 32, 318–325
Lunder, M., Bratkovic, T., Urleb, U., Kreft, S., & Strukelj, B (2008) Ultrasound in phage
display: A new approach to nonspecific elution Biotechniques, 44, 893–900
Menchicchi, B., Fuenzalida, J P., Bobbili, K B., Hensel, A., Swamy, M J., & Goycoolea, F.
M (2014) Structure of chitosan determines its interactions with mucin Biomacromolecules, 15(10), 3550–3558
Polonelli, L., Giovati, L., Magliani, W., Conti, S., Sforza, S., Calabretta, A., et al (2011) Vaccination of lactating dairy cows for the prevention of aflatoxin B1 carry over in milk Public Library Of Science, 6, e26777
Quemeneur, F., Rammal, A., Rinaudo, M., & Pepin-Donat, B (2007) Large and giant vesicles decorated with chitosan: Effects of pH, salt or glucose stress and surface adhesion Biomacromolecules, 8, 2512–2519
Ramada, J S., Becker-Finco, A., Minozzo, J C., Felicori, L F., de Avila, R A M., Molina, R., et al (2013) Synthetic peptides for in vitro evaluation of the neutralizing potency
of loxosceles antivenoms Toxicon, 73, 47–55
Recillas, M., Silva, L L., Peniche, C., Goycoolea, F M., Rinaudo, M., & Arguelles-Monal,
W M (2009) Thermoresponsive behavior of chitosan-g-N-isopropylacrylamide co-polymer solutions Biomacromolecules, 10, 1633–1641
Rodrigues, S., Dionísio, M., Remuñán López, C., & Grenha, A (2012) Biocompatibility of chitosan carriers with application in drug delivery Journal of Funcional Biomaterials,
3, 615–641
Sagona, A P., Grigonyte, A M., MacDonald, P R., & Jaramillo, A (2016) Genetically modified bacteriophages Integrative Biology, 8, 465–474
Scott, J K., & Smith, G P (1990) Searching for peptide ligands with an epitope library Science, 249, 386–390
Silva, C A., Nobre, T M., Pavinatto, F J., & Oliveira, O N (2012) Interaction of chitosan and mucin in a biomembrane model environment Journal of Colloid and Interface Science, 376(1), 289–295
Sun, B., & Xia, T (2016) Nanomaterial-based vaccine adjuvants Journal of Materials Chemistry B, 4, 5496–5509
Thirumala‐Devi, K., Miller, J S., Reddy, G., Reddy, D V R., & Mayo, M A (2001) Phage-displayed peptides that mimic aflatoxin B1 in serological reactivity Journal of Applied Microbiology, 90, 330–336
Tsapis, N., Bennett, D., Jackson, B., Weitz, D A., & Edwards, D A (2002) Trojan par-ticles: Large porous carriers of nanoparticles for drug delivery Proceedings of the National Academy of Sciences, 99, 12001–12005
Van der Lubben, I M., Verhoef, J C., Borchard, G., & Junginger, H E (2001) Chitosan for mucosal vaccination Advanced Drug Delivery Reviews, 52(2), 139–144
Vila, A., Sánchez, A., Janes, K., Behrens, I., Kissel, T., Jato, J L V., et al (2003) Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice European Journal of Pharmaceutics and Biopharmaceutics, 57, 123–131
WHO (1993) World health organization: some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins Monograph 56 France: International Agency for Research on Cancer Lyon
WHO (2002) World health organization: Some traditional herbal medicines, some mycotoxins, naphthalene and styrene Monograph 82 France: International Agency for Research on Cancer Lyon
WHO (2005) World health organization: Public health strategies for preventing aflatoxin exposure Geneva, Switzerland: WHO
Wang, Y., Wang, H., Li, P., Zhang, Q., Kim, H J., Gee, S J., et al (2013) Phage-displayed peptide that mimics aflatoxins and its application in immunoassay Journal of Agricultural and Food Chemistry, 61, 2426–2433
Wild, C P., Miller, J D., & Groopman, J D (2015) Mycotoxin control in low-and middle-income countries France: International Agency for Research on Cancer Lyon
Wilkinson, J., Rood, D., Minior, D., Guillard, K., Darre, M., & Silbart, L K (2003) Immune response to a mucosally administered aflatoxin B1 vaccine Poultry Science,
82, 1565–1572
Yamamoto, H., Kuno, Y., Sugimoto, S., Takeuchi, H., & Kawashima, Y (2005) Surface-modified PLGA nanosphere with chtiosan improved pulmonar delivery of calcitionin
by muoadhesion and opening of the intercelular tight junctions Journal of Controlled Release, 102, 373–381
Yoo, M K., Kang, S K., Choi, J H., Park, I K., Na, H S., Lee, H C., et al (2010) Targeted delivery of chitosan nanoparticles to Peyer’s patch using M cell-homing peptide se-lected by phage display technique Biomaterials, 31, 7738–7747