Development of a Ferritin-Based Nano-Allergen and Its Immunological Effects in vitro and in vivo.

INTRODUCTION

We aimed to develop recombinant allergens on the nanoscale by using self-assembly property of ferritin and to investigate their immune response and protective effect.

METHODS

The cDNA encoding Ftn-Blo t 2 was synthesized and cloned into pET-21a (+) vector, then expressed in BL21 (DE3). Recombinant proteins (rBlo t 2, rFtn, and rFtn-Blo t 2) were purified and their immunoreactivity confirmed for immunoreactivity by IgE-ELISA and Western blot. In vitro, the immunogenicity of rFtn-Blo t 2 was assessed using BEAS-2B human bronchial epithelial cells by measuring epithelial cytokine responses. In vivo, a murine airway inflammation model was established via sensitization and challenge with crude extract, followed by treatment with rFtn-Blo t 2 to investigate its effects on airway inflammation, Th1/Th2 cytokine profiles, and allergen-specific antibody production.

RESULTS

The average diameter of rFtn and rFtn-Blo t 2 particles are (11.24 ± 1.31) nm and (16.00 ± 1.59) nm. The IgE- binding rates of rFtn-Blo t 2 and rBlo t 2 were 62.5% (15/24) and 58.3% (12/24), respectively. The expressions of IL-25, IL-33, and TSLP increased by 3.29, 3.43, and 2.22 folds after the addition of rFtn-Blo t 2 in co-culture with BEAS-2B. Mice challenged with rBlo t 2 had lower levels of cytokines IL-13 and IL-4 but higher levels of IFN-γ and TGF-β in alveolar lavage fluid compared to controls (p < 0.05). HE staining showed that compared with rBlo t 2 group, the inflammation level in lung tissue of rFtn-Blo t 2 group was reduced.

DISCUSSION

A nano-scale allergen was successfully developed, providing a concept, a platform, and a paradigm for the construction of nano-scale allergens.