Encapsulation of Allergens into Chitosan-Alginate Nanoparticles Prevents IgE Binding
E. Kashirina1, P. Reshetov1, L. Alekseeva1, V. Berzhets2, D. Ryazantsev1, V. Zubov1, D. Chudakov1, and E. Svirshchevskaya1*
1Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
2Mechnikov Scientific Research Institute of Vaccines and Sera, Russian Academy of Medical Sciences, Moscow, Russia
Allergens used for allergen specific immunotherapy (ASIT) can induce side effects due to the binding of IgE immobilized on mast cells to allergens. To prevent IgE binding, recombinant allergens from house dust mites (HDM) Dermatophagoides farinae Der f 2 or Aspergillus fumigatus (AF) fungi Asp f 3 were encapsulated into nanoparticles (NPs) developed from biodegradable polymers chitosan and alginate. Core NPs 400-440 nm in diameter and zeta potential +8 mV were prepared from amphiphilic chitosan derivative N-lauryl-N’-succinoylchitosan (LSC). Allergens Der f 2 or Asp f 3 were introduced during core NP formation. To mask allergens additionally, allergen loaded core NPs were coated by alginate shell. As a result of polyelectrolyte interaction between positively charged LSC and negatively charged alginic acid core-shell type NPs were formed which were 450-550 nm in diameter and zeta potential –13 to –20 mV. We demonstrated that the encapsulation of allergens into core NPs significantly decreased IgE reactivity while core–shell NPs masked the allergens completely, as was shown using IgE positive sera from patients allergic to HDMs or AF. Injection s.c. of both core and core-shell NPs into mice induced allergen specific IgG response comparable to free allergens. We concluded that encapsulation of allergens into coreshell NPs could increase the safety and preserve the efficacy of preparations for ASIT.
Keywords: Allergy; Specific Immunotherapy; House Dust Mites; Aspergillus fumigatus Fungi; Chitosan; Alginate, Nanoparticles,
ASIT: Allergen-Specific Immunotherapy;
DLS: Dynamic Light Scattering;
ELISA: Enzyme Linked Immunosorbent Assay;
HDMs: House Dust Mites;
AF: Aspergillus fumigatus fungi;
LSC: N-lauryl-N’-succinoyl chitosan
DCC: N, N’- Dicyclohexylcarbodiimide;
Type I allergic reactions are characterized by the formation of E class antibodies to harmless microorganisms that enter the body from the environment, such as tree or grass pollen, fragments of domestic microorganisms, food, etc. For more than 100 years, the only method to reduce allergic symptoms was allergen specific immunotherapy (ASIT), which consists of the long-term courses (up to 3 years) of subcutaneous (s.c.) administration of allergen extracts in very low doses . S.c. administration of allergens to allergic patients is associated with the development of IgE mediated reactions, which can reach the level of anaphylactic shock. Therefore, the main aim in the design of new formulations for ASIT is to prevent the recognition of allergens, incorporated into vaccines, by IgE immobilized on the mast cells in order to avoid the risk of adverse reactions.
Modification of allergens such as treatment with glutaraldehyde (change of protein conformation), the use of mutant forms of allergens (removal of some IgE binding sites), conjugation of allergens with polymers (masking of some antigenic sites), etc are well known approaches to reduce allergenic and protect immunogenic properties of allergens [2–4]. Modified allergens with reduced ability to interact with IgE are much safer and can be used at doses several times higher than those of the standard allergen extracts.
At the moment, intensive research is being conducted to produce anti-allergic vaccines based on nanotechnology including chitosan nanoparticles (NPs) [5, 6]. Chitosan is a natural biocompatible and biodegradable polymer with adjuvant activity. Encapsulation of HDM allergens into chitosan-based NPs was carried out by Liu Z. et al [7–9] where the effects of Der f 2 47–67 peptide  or allergen extract [8, 9] incorporated into chitosan NPs were studied. Administration of allergen containing NPs via intraperitoneal (peptide) , intranasal or sublingual routes (extract) [8, 9] reduced symptoms in the mouse model of HDM allergy. However, it is not clear whether the encapsulation is sufficient to prevent human IgE recognition. Earlier we have shown that incorporation of HDM allergens into single polymer NPs is not sufficient to completely prevent IgE binding and additional shell is required for safe allergen packaging .
The aim of this study was the production and characterization of core-shell chitosan-alginate NPs containing HDM major allergen Der f 2 or AF major allergen Asp f 3. Allergens were chosen to be most often involved in IgE recognition in allergy induced by HDM  and allergic bronchopulmonary aspergillosis .
Materials and Methods
Medium molecular weight (MW) chitosan with deacetylation degree 0.94 (Sigma); low MW sodium alginate from Macrocystis pyrifera (1 cps viscosity) (Sigma); N,N’-dicyclohexylcarbodiimide (DCC) (Sigma); 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) (Pierce); succinic anhydride (Pierce); N-hydroxysuccinimide (Reanal); lauric acid (Acros Organics); fluorescein isothiocyanate (FITC) and rhodamine B (Rho) (Sigma) were used.
N-hydroxysuccinimide ester of lauric acid
Lauric acid (192 mg, 1 mmol) and DCC solution (193 mg, 1 mmol) in 3 mL of dry ethyl acetate were added to N-hydroxysuccinimide solution (115 mg, 1 mmol) in 5 mL dry ethyl acetate under stirring and the reaction was stirred for 12 h at RT. The precipitate of N,N’-dicyclohexylurea was separated by filtration and the filtrate was evaporated to dryness. The residue was recrystallized from ethanol and air dried. White crystals of N-hydroxysuccinimidyl ester of lauric acid (yield 50 mg, 80%) were obtained.
N-hydroxysuccinimide ester of lauric acid (22.5 mg, 0.1 mol) in 10 mL of methanol was added to the chitosan solution (100 mg, 2.5 μmol) in 10 mL of 2% acetic acid and 30 mL methanol, reaction mixture was stirred for 12 h at RT. Methanol was evaporated and the aqueous solution was dialyzed against 0.1% acetic acid and freeze dried; light white powder of LC (yield: 85 mg; 85%) was obtained.
N-lauryl-N’-succinoyl chitosan (LSC)
Succinic anhydride (31.8 mg, 100 pmol) was added to the LC solution (80 mg, 2 mol) in 8 mL of 2% acetic acid and 32 mL of methanol and the reaction mixture was stirred for 12 h at RT. Methanol was evaporated and the aqueous solution was dialyzed against 0.1% acetic acid and freeze dried; a light white powder of LSC (yield 65 mg, 81%) was obtained.
Recombinant E. Coli Der f 2 was obtained as previously described . Asp f 3 was a kind gift of Dr.V.P. Kurup (USA) .
HDM and AF patients
Sera from children allergic to HDMs (n=8) or AF (n=3) were collected in Mechnikov Scientific Research Institute of Vaccines and Sera (Moscow, Russian Federation) before ASIT and with the written agreement of patients’ parents. IgE specificity was identified by RIDA®AllergoScreen (Germany).
Production of encapsulated allergens
Production of core LSC NPs
EDC (5.76 mg, 30.3 μmol) was added to the LSC solution (10 mg, 0.17 μmol) in 3 mL PBS, pH 7.2, and a solution of recombinant
Der f 2 or Asp f 3 proteins (1 mg of each protein in 200 μL PBS) was added drop wise after 10 min; pH 7.4 of the reaction mixture was maintained with 5M sodium bicarbonate. The reaction was conducted for 4 h at room temperature at stirring. LSC NPs were formed during the dialysis against PBS for 4 h. Completeness of the reaction was verified by 10% PAGE indicating a 100% yield during the condensation stage (Fig.1B). An aliquot of the suspension was freeze dried and weighed (yield: 9.0 mg; 90% for LSC) to determine the yield of the conjugate.
Rhodamine B labeling of Der f 2 and Asp f 3
Fluorescently labeled proteins were prepared to estimate the amount and to visualize the proteins included into the core NPs. To prepare these proteins, EDC activated rhodamine B (Rho) solution (0.4 μg, 1 μmol) in 0.4 mL of water was added to 0.6 mL of Der f 2 or Asp f 3 protein solution (500 μg) in PBS and stirred for 12 h. The excess of the dye was removed by overnight dialysis against PBS. The resulting labeled proteins were used for the synthesis of the core NPs as described above. The amount of proteins bound to the particles was determined using GlomaxMulti spectrofluorimeter (Promega, United States) with a 525-nm filter. Protein concentrations were determined using free Rho-labeled protein titration curve. LSC to protein ratio was 10-15 to 1.
Core NPs containing either Der f 2 or Asp f 3 (5 mL) was added slowly with vigorous stirring to 10 mL sodium alginate solution in PBS (5 mg/ml) and stirred for 30 min; the formation of opalescent NP suspension was observed. NPs were purified from unbound alginate by centrifugation at 14000 rpm for 20 min. Approximate ratio of alginate/LSC/protein was 5/10/1. For the visualization core-shell NPs were labeled by FITC as described earlier . For some experiments Asp f 3 or Der f 2 core-shell NPs were freeze dried, reconstituted with PBS and used to immunize mice.
1H-NMR spectra of the initial chitosan and its derivatives were obtained using Bruker DRX 500 spectrometer (500 MHz) (United States) in 0.01 M DCl/D2O at 30oC. The degree of substitution (succinoyl/lauryl) was calculated relatively to the integrated intensity of proton signals of N-succinoyl and N-lauryl radicals to the signal of protons in 2C; that is, 12 fatty acid residues and 30 succinoyl residues per 100 monosaccharide units for LSC.
Dynamic light scattering (DLS)
The average size of core and core-shell NPs was determined using 90 Plus Particle Size Analyzer (Brookhaven, United States) in water (25.0 ± 0.1oC) at a scattering angle of 90o and wavelength of 661 nm using Big Particle Sizing Software. Zeta potential of NPs was determined in 10 mM KCl solution using identical Big Pal Zeta-Potential analyzer hard-ware and software.
Enzyme Linked Immunosorbent Assay (ELISA)
IgE and IgG specific ELISA
Der f 2, Asp f 3 recombinant proteins, core or core-shell NPs in PBS in concentration corresponding to 5 μg/mL of protein in 100 μL/well were loaded onto 96-well plates (Nunc, Denmark) and incubated overnight at 4oC. Plates were then washed 3 times with PBS containing 0.05% Tween-20, blocked with 1% bovine serum albumin (Serva, Germany) in PBS (PBA) and sera of patients allergic to HDMs or AF were added at different dilutions. Plates were incubated for 2 h at RT, washed as described above, and mouse IgG1 anti-human-IgE antibodies in PBA (100 μL, 2 μg/mL, SantaCruz, United States) were added for 1 h followed by goat-anti-mouse IgG-HRP conjugate for 1 h at RT. After four washes, 3,3’,5,5’-tetramethyl-benzidine (TMB) solution was added. After 15–20 min, the reaction was stopped by adding 10% sulfuric acid and the plates were analyzed using plate spectrophotometer Titertek (United Kingdom) at 450 nm. Sera from mice immunized with NPs or rabbits immunized with recombinant allergens Der f 2 or Asp f 3 were tested in the same way using goat-anti-mouse or goat-anti-rabbit IgG-HRP conjugates.
To estimate adhesion of NPs to plastic, free recombinant proteins Der f 2, Asp f 3, core, or core-shell NPs were loaded at 5 μg/mL of protein in 100 μL/well onto 96-well plates (Nunc, Den-mark) and incubated overnight at 4oC. High titers rabbit anti Der f 2 or Asp f 3 sera were used to compare antigen recognition in free or encapsulated forms as described above.
Phagocytosis of NPs was analyzed using mouse macrophage cell line J774. Cells were cultivated in RPMI-1640 medium, supplemented with antibiotics, L-glutamine, and 7% fetal serum (all from PanEco, Moscow). For the analysis J774 cells were trypsinized, 104 cells were seeded in 200 μL onto sterile cover glasses until complete adherence. NPs in PBS (10 μL) were added to the cells and incubated overnight at 37oC under 5% CO2. Nuclei were stained with Hoechst 33342 (Sigma). Co-localization with lysosomes was performed using LyzoTrackerGreen (Molecular Probes, United States) respectively. The unbound particles and dyes were washed out using a nutrient medium; cells were fixed with 4% paraformaldehyde, washed 3 times in PBS, and embedded in Mowiol 4.88 (Calbiochem, Germany). Samples were analyzed using Nikon TE 2000 Eclipse confocal microscope (Japan). To estimate in vivo traffic of NPs, BALB/ c mice (n=2) were injected s.c. into the hind footpad with 50 μL (2 μg of protein) of Rho-labeled core or core-shell NPs without adjuvant. A week later mice were sacrificed and the adjacent popliteal lymph nodes were collected. Lymph nodes were fixed in 4% paraformaldehyde, imbedded into cryomedium Killik (Bio-Optica, Italy) and sectioned using Cryotome™ FSE Cryostats (Thermo Scientific, USA). Sections were stained with Hoechst 33342 (Sigma), macrophages were stained with anti-CD11b-FITC antibody (BioLegend) and analyzed using
In vivo experiments
Immunization of rabbits
Rabbits purchased from Stolbovaya farm (Moscow region) were immunized intramascular in the hind leg with 50 μg of Der f 2 or Asp f 3 admixed 1:1 with Complete Freund Adjuvant (Sigma). A month later immunization was repeated using the same amount of antigens admixed with Incomplete Freund Adjuvant. Sera were collected at 1 and 2 weeks after the last immunization. All animal studies were approved by the Ethical Commission (IACUC) of the Institute.
Immunization of mice
BALB/c mice were purchased from Stolbovaya farm (Moscow region). Mice (n=3 per group) were immunized twice with a week interval s.c. in the tail base with 50 μL of NPs or free allergens (total dose 20 μg of proteins). In some experiments as pointed in the Result section free Asp f 3 was used with Alum (Termo Scientific, USA) otherwise free allergens were injected in PBS. As an alternative, some mice (n=3 per group) were immunized intranazally (i.n.) with core-shell NPs. To do it, mice anaesthetized with xylazine hydrocholide were kept in the upright position and instilled with 25 μL of NPs once in three days alternating the nostrils, totally 5 times (25 μg total dose). Blood was collected from orbital sinuses 1 and 2 weeks after the last immunization. IgG was determined by ELISA.
Statistical analysis was performed using Microsoft Excel Software. Differences were considered statistically significant if the probability value of the Student’s t-test null hypothesis was p < 0.05.
Results and Discussion
Preparation and Characteristics of allergen loaded core– shell nanoparticles
Chitosan modification was carried out in order to optimize the hydrophilic–lipophilic and acidic-basic properties. The introduction of lauryl fatty acid residues into chitosan bone enables the formation of NPs in aqueous medium due to hydrophobic interchain interactions while the presence of dicarbonic acid residues increases the solubility of chitosan at physiological pH. As a result we obtained lauryl-succinoyl-chitosan (LSC) with degree of substitution 12% for lauryl and 30% for carboxyl residues as was determined by 1H-NMR (Fig. 1A).
Figure 1. Characteristics of NPs. A. 1H-NMR spectra of LSC. Proton chemical shifts are shown relative to tetramethylsilane: 2.54 ppm (CH2)2 (succinoyl) (1); 2.04 ppm N-acetyl (2); 1.21 ppm (CH2)10 (lauryl) (3). B. PAGE analysis of 18 kDa Asp f 3 (1); 14 kDa Der f 2 (2), Asp f 3 core NPs (3); Der f 2 core-shell NPs (4). C. Schema of core-shell NP formation. D-E. DLS analysis of Asp f 3 core (D) and core-shell NPs (E). F-G. Confocal images of Der f 2 core NPs (F) and Der f 2 core-shell NPs.
This gave zeta-potential around +10 mV and the ability to form NPs spontaneously at neutral pH. LSC was used as a matrix to pack recombinant allergens Der f 2 or Asp f 3 via irreversible carbodiimide mediated cross-binding to prevent allergen leakage. Gel electrophoresis demonstrated complete binding of proteins to the matrix (Fig.1B). Allergen-LSC conjugates formed core NPs via self-assembly during dialysis as shown schematically in Fig.1C. Core NPs were slightly positively charged (ζ˷ +8 mV) and had approximately 400 nm in diameter as determined by DLS (Fig. 1D and E).
Rho-labeled Asp f 3 and Der f 2 proteins (Rho-Asp f 3; Rho-Der f 2) were used to estimate the amount of protein incorporated into core NPs. Direct measurement of unlabeled protein concentrations in NPs failed possibly due to LSC interference and low protein content in NPs. Concentration was determined using free Rho-Asp f 3 or Rho-Der f 2 titration curves and was around 200 μg/mL. The amount of bound protein was similar (˷95%) to the amount added to the reaction, confirming the completeness of the reaction. The dry weight of samples was used to determine the yield of NPs. LSC/protein ratio was 10- 15 to 1 depending on the batch.
Allergens incorporated into core NPs could possibly be partially exposed on NP surfaces as depicted in Figure 1C. These exposed residues of allergens were covered by alginate shell formed by electrostatic interactions between positively charged LSC and negatively charged alginic acid (Fig. 1C). The amount of alginate was selected to form negatively charged core-shell NPs (ζ˷-10 to - 15 mV). As estimated by freeze drying and weighing, an approximate ratio of alginate/LSC/protein was 5/10/1 both for Asp f 3 and Der f 2 allergens. The diameter of core-shell NPs slightly increased from 400 to 450 nm (Fig. 1, D-E) showing a dense packing of polymers.
To visualize core and core-shell NPs a batch of each NPs was FITC labeled. Confocal images demonstrated different forms of core and core-shell NPs: round for core NPs and irregular roundish for core-shell ones (Fig.1F-G).
IgE binding by encapsulated allergens
IgE binding was estimated by ELISA using sera from HDM (n=8) or AF (n=3) sensitized children as verified by commercial RIDA®AllergyScreen test. ELISA is based on the binding of serum immunoglobulins to the antigens immobilized on the solid surface (sorbent assay). However chitosan-alginate NPs could possibly poorly bind the plates. So we had to be sure that NPs are immobilized on the ELISA plates. To verify this, rabbits were immunized with recombinant Der f 2 or Asp f 3 and high titer sera were used to compare the amount of allergens immobilized. Earlier we have shown that high affinity IgG can bind target antigens even when they are exposed inside protein NPs . Indeed, both free and encapsulated Asp f 3 were equally recognized by high affinity rabbit anti-Asp f 3 IgG (Fig. 2A). The same was found for Der f 2 NPs (data not shown). These results demonstrate that polymeric shell does not decrease high affinity IgG binding.
Figure 2. Recognition of encapsulated allergens by IgG and IgE. A. Verification of NPs adhesion to plastic. Recognition by high titer rabbit anti-Asp f 3 specific serum of free or encapsulated Asp f 3. B-C. Recognition of free or encapsulated allergens by IgE in sera from AF (B) or HDM (C) sensitive children; 1:100 dilution. D-F. Titration curves of sera from HDM patients on free Der f 2 (D); Der f 2 core NPs (E); or Der f 2 core-shell NPs (F).
Affinity of IgE is rather low in comparison with IgG. It is known that IgE recognize mostly conformational epitopes while high affinity IgG – liner ones [16-18]. ELISA analysis of IgE binding demonstrated that double encapsulation was sufficient to completely mask IgE epitopes of both Asp f 3 (Fig. 2B) and Der f 2 (Fig. 2C-F) allergens while LSC coating significantly (p<0.001) but not completely decreased IgE binding. These results also show that IgE has low affinity to allergens, at least for anti HDM or AF IgE used. Thus, encapsulated allergens can be used at much higher concentrations for ASIT. However, allergens
packed into a polymeric matrix can loose immunogenic properties.
Immunogenic properties of encapsulated allergens Asp f 3 and Der f 2
Humoral immune response is initiated by the antigen phagocytosis by resident tissue macrophages which transport the antigen to the adjacent lymph node. The B-cell switch from IgM to IgG is mediated by T helper cells [19-20]. For the effective antigen presentation to T cells the antigen should be processed via endosomal-lysosomal pathway . Phagocytosis and endosomal processing of NPs was studied in macrophage- like murine cell line J744. Time dependent analysis demonstrated that NPs were transported inside the cells rather slowly and were mostly found at the cell surface after 6 h incubation (supplemental Fig.A). However, after 24 h incubation both Asp f 3 (Fig.3A) and Der f 2 (Fig.3B) core-shell NPs were found inside macrophage lysosomal compartment as was estimated by co-localization experiments with lysosomal tracker.
Figure 3. In vitro and in vivo traffic of allergen loaded core-shell NPs. A-B. In vitro 24 hrs traffic of rhodamine labeled (red) Der f 2 core-shell NPs (A) and Asp f 3 core-shell NPs (B) in macrophage cell line J774. Lysosomes are stained in green; nuclei – in blue. Scale bar corresponds to 15 μm. C-D. Cryosections of politeal lymph nodes of mice injected with rhodamine labeled Der f 2 core-shell NPs (C, red) and Asp f 3 core-shell NPs (D, red) into the hind footpad 3 days before. Macrophages are stained with anti-CD11b-FITC antibodies (C, green), nuclei – with Hoechst (C-D, blue). Scale bar corresponds to 50 μm.
To translate in vitro results into in vivo model, mice were injected s.c. with Rho-Asp f 3 or Rho-Der f 2 core-shell NPs and a week later adjacent popliteal lymph nodes were collected and analyzed. We demonstrated that both allergens were detected in the lymph nodes (Fig.3B-C) and were co-localized with CD11b positive macrophages (Fig.3B).
To compare mounted IgG responses, mice were s.c. immunized by free or encapsulated Asp f 3; PBS or empty NPs. Free Asp f 3 was used mixed with Alum adjuvant (Asp f 3–Alum). Immunization was repeated 2 times with a week interval. Sera were analyzed by ELISA against recombinant Asp f 3. We demonstrated that Asp f 3–Alum induced the highest IgG response followed by Asp f 2 core and core-shell NPs (Fig. 4A). Immunization with free Der f 2 without Alum and encapsulated Der f 2 induced comparable IgG responses (Fig. 4B). There were no differences in IgG responses induced by core or core-shell allergen loaded NPs. Empty polymeric NPs did not induce allergen specific IgG as it was well expected. These results demonstrate high immunogenicity of allergens encapsulated into chitosan-alginate polymeric NPs.
Figure 4. Immunogenic properties of allergen loaded NPs. A. IgG response to free Asp f 3, Asp f 3 loaded core or core-shell NPs, and control empty core or core-shell NPs after 2 s.c. injections. B. IgG response to free Der f 2, Der f 2 loaded core or core-shell NPs after 2 s.c. injections. C-D. Induction of IgG response in mice immunized with Asp f 3 (C) or Der f 2 (D) core-shell NPs via s.c. (2x) or i.n. (5x) routs before and after NPs lyophilization and reconstitution.
The role of IgG in the protection from allergy is still a matter of debate. There are numerous studies demonstrating the roles of Th1 or Treg in hyposensibilization [22-23]. At the same time successful ASIT is always associated with the allergen-specific IgG4 increase [22-23]. Th1 and Treg analysis was beyond the scope of our study and can be conducted later. We believe that high affinity humoral IgG response is essential for the protection.
Allergens during ASIT can be introduced by different routes such as by subcutaneous injection, per oral, sublingual, intranazal (i.n.), or transepidermal ones [22-25]. Nonparental treatment has many advantageous over s.c. parental therapy due to the avoidance of injections which is especially important for children; better compliance for patients; less chances of adverse reactions; less attendance of therapists. However nonparental medication has to bypass the epithelial barriers and thus can be less effective in the treatment.
Chitosan and chitosan NPs were shown to open tight junctions of epithelial cells [26-28]. So, we tried to deliver allergens via i.n. route. In a parallel we also estimated whether dried and reconstituted encapsulated allergens preserve immunogenic activity. Mice were immunized with control or dried and reconstituted Der f 2 or Asp f 3 core-shell NPs via s.c. (2 times) or i.n. (5 times) routes and allergen specific IgG was estimated by ELISA. We demonstrated that both routes effectively induced allergen specific IgG response (Fig.4 C-D). I.n. protocol was less efficient than s.c. one and lyophilization decreased the activity approximately 1.3 fold.
Allergic diseases affect mostly young people decreasing the quality of their lives and resistance to various infections. ASIT is the only pathophysiological treatment for allergy preventing its progression however it is often not chosen as a remedy both because allergy is not a life threatening disease and the availability of anti-histamine quick rescue treatment. Development of effective, safe, and short treatment for allergy will stimulate adherence of allergic people to ASIT improving not only their condition but also saving a vast majority of money used by health care system. Summarizing our results, we showed a pathway how to develop such vaccines. Encapsulation of any allergens, both recombinant or native extracts, into biodegradable carrier which can mask IgE epitopes, can significantly reduce the risk of side effects associated with ASIT and increase the immunogenic dose. In this work chitosan-alginate NPs were used as a carrier and shelter of allergens which significantly decreased the recognition of the proteins by IgE antibodies, but did not affect the phagocytosis and processing of NPs by macrophages. Moreover, encapsulated allergens were presented by macrophages to T and B cells leading to the formation of allergen specific IgG. We showed that such NPs can be used for s.c. or i.n. ASIT at higher concentrations than conventional preparations leading to a shorter therapeutic course. Chitosan-alginate NPs can be freeze dried and reconstituted with significant antigenic activity preserved which makes this approach easily translated from bench-to-bedside. However, their protective anti-allergic activity should be demonstrated in murine models of allergy.
This work was supported by the Program “Nanotechnologies & Nanomaterials” of the Russian Academy of Sciences; by the Russian Fund for Basic Research, grants #12-04-01792а, #16- 03-01093, and Russian Scientific Fund, #16-14-00136 .
Cite this article: E. Svirshchevskaya. Encapsulation of Allergens into Chitosan-Alginate Nanoparticles Prevents IgE Binding. J J Vaccine Vaccination. 2015, 1(3): 012.