AL, HW and LR performed the animal experiments. a diameter of 200?nm showed a positive zeta potential. The coiled-coil conjugation of Bet v 1 to the surface of liposomes resulted in about a 15-fold lower allergenicity than soluble Bet v 1 as judged by RBL assays. Moreover, the nanoparticles induced Bet v 1-specific IgG1/IgG2a responses in mice that were several orders of magnitude higher than those induced by alum-adsorbed Bet v 1. This strong humoral response was accompanied by a relatively strong IL-10 induction upon PBMC stimulation with Bet v 1. In conclusion, their hypo-allergenic properties, combined with their capacity to induce a strong humoral immune response and a relatively strong IL-10 production, makes these allergen-covered cationic liposomes a promising alternative for aluminum salt-adsorption of allergen currently used in SCIT. Keywords: aluminum, bet v 1, hypo-allergenic, liposomes, mice Introduction Subcutaneous allergen immunotherapy (SCIT) has been used to treat allergies for more than 100 years (1). The treatment commonly consists of monthly subcutaneous injections of allergen extracts for 3 to 5 5 years to achieve optimal therapeutic effect. Therapy adherence is relatively low because of this long duration and the allergic side-effects that can occur (2). Often, aluminum hydroxide (alum) or aluminum phosphate is used as adjuvant for SCIT. Although alum has been reported to skew towards T AZD1208 HCl helper (Th) 2 immune responses (3), during SCIT it has been shown to result in a more mixed Th1/Th2 cytokine response in combination with production of interleukin (IL)-10 by regulatory T- and B-cells (4, 5). Most importantly, these regulatory B-cells also produce the required protective allergen-specific immunoglobulin (Ig) G4 antibodies. Alum has a long history of safe use in vaccines for infectious diseases but also in SCIT (3). Nevertheless, there is some concern with respect to long-term exposure to alum during allergen immunotherapy (AIT), particularly in a pediatric setting (6). Although as yet there is little or no evidence to support aluminum-associated pathology during AIT, a search for good alternatives may nevertheless be warranted. Besides directing the immune response, alum also serves as a depot for adsorption of allergens, partially shielding them from IgE antibodies and thereby reducing the risk of allergic side-effects (7). In recent years, different types of nanoparticles have drawn attention to serve as effective vaccine delivery systems (8C10). Liposomes are amongst the most promising nanoparticles to replace alum (11, 12). Liposomes consist of one or more lipid bilayers with an aqueous core and are a versatile delivery system and adjuvant for vaccines (11C13). Antigens can be adsorbed to the lipid bilayer (14), incorporated in the lipid bilayer (15), or encapsulated in the aqueous core of the vesicle (16, 17). Recently, we described a novel antigen-anchoring method based on the interaction between two complementary CC formation resulted in strong CD4+ T-cell proliferation and production of both interferon gamma (IFN-a poly(ethylene glycol) spacer to enable anchoring of the peptide into the lipid bilayer of liposomes and was synthesized as AZD1208 HCl described elsewhere (18). In short, Fmoc-NH-PEG4-COOH was coupled to resin-bound (KIAALKE)4 in the presence of DIPEA (5 eq.) and HATU (2.5 eq.) for 2.5?h. Fmoc-deprotection was done with 20% piperidine in DMF and the reactive amine was coupled to amino-cholestene hemi-succinate AZD1208 HCl (1.05 eq.) in the presence of DIPEA (5 eq.) and HATU (2.5 eq.) for 4?h at room temperature. The peptide was cleaved from the resin with a mixture of TFA:TIPS:water (95:2.5:2.5?v/v/v) and precipitated in Nr4a3 ice-cold diethyl ether. Crude AZD1208 HCl peptides were purified using a Shimadzu RP-HPLC system comprising two LC-8A pumps and an SPD-10AVP UV-Vis detector equipped with a Kinetic Evo C18 column. A gradient of 20%C80% B, (where B is 1% (v/v) TFA in ACN, and A is 1% (v/v) TFA in water) with a flow rate of 12?ml/min was used. Collected fractions were measured on a LC-MS system (Thermo Scientific TSQ quantum access MAX mass detector connected to an Ultimate 3,000 liquid chromatography system fitted with a 50??4.6?mm Phenomenex Gemini 3?BL21 (DE3) cells for protein production. The harvested cell pellets were disrupted by sonication and cellular debris was removed by centrifugation. The pepE-Bet v 1 protein was purified from the supernatant by affinity purification using cross-linked agarose beads functionalized with PepK. Bound pepE-Bet v 1 was eluted by lowering the pH to 2.5 to unfold the pepE/pepK coiled coil. Elution fractions containing pepE-Bet.
AL, HW and LR performed the animal experiments