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

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.

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 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: rilton@ufpr.br, rilton@quimica.ufpr.br (R.A de Freitas), lmalvarenga@ufpr.br (L.M Alvarenga) https://doi.org/10.1016/j.carbpol.2017.12.063 Received 29 September 2017; Received in revised form 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 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 (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 64 Carbohydrate Polymers 185 (2018) 63–72 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 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 °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 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, °C) and analyzed regarding their binding to specific antibody Microtitration plates (Nunc-Imuno, MaxiSorp F96, Nunc, Roskilde, Denmark) were coated for 16 h at °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 h at 37 °C A wild type phage is identical to phage clones present in the libraries, but 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 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 × 1013 pfu mL−1 and a final solution was obtained after dilution in 10 mL ultrapure water to a concentration of × 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 65 Carbohydrate Polymers 185 (2018) 63–72 C.Y.T de Andrade et al acetic acid/sodium acetate buffer, mg mL−1, pH 4.6, under continuous mild stirring After complete mixture the solution was maintained in agitation for at least 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 and 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 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 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 h and labeled with Alexa-fluor 633-conjugated antibody anti-IgG murine (Thermo Scientific, Waltham, USA) for 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 mg mL−1 of chitosan) in 10 μL of PBS were administered in the animal nostrils 66 Carbohydrate Polymers 185 (2018) 63–72 C.Y.T de Andrade et al Fig 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 67 Carbohydrate Polymers 185 (2018) 63–72 C.Y.T de Andrade et al Fig 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 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 68 Carbohydrate Polymers 185 (2018) 63–72 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 (< 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 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 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 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) 69 Carbohydrate Polymers 185 (2018) 63–72 C.Y.T de Andrade et al Table Apparent hydrodynamic radius and ζ-potential of phages and chitosan-coated phages at different pH values All results represent the average of 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 ± 89 ± 15 345 ± 26 116 ± 250 ± 80 444 ± 150 −33 ± +35 ± +40 ± −80 ± 10 −10 ± +20 ± 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, 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 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 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 70 Carbohydrate Polymers 185 (2018) 63–72 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 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 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 online version, at https://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 reagents 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 dermonecrotic activity of Loxosceles intermedia spider venom and their use in a specific 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