Dongmei Zhou,1,* Yaning Ren,1,* Ying Zhou,2 Yuanfen Liao,1 Qi Cheng,1 Jinni Chen,3 Cunyin Yuan,1 Dan Zeng,4 Yubao Cui1 

1Clinical Research Center, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, 214023, People’s Republic of China; 2Department of Pediatrics Laboratory, The Affiliated Children´s Hospital of Jiangnan University, Wuxi, 214023, People’s Republic of China; 3Department of Respiratory, Hainan Women and Children’s Medical Center, Affiliated Pediatrics Clinical College of Hainan Medical University, Haikou, 570100, People’s Republic of China; 4Department of Allergy, Chongqing General Hospital, Chongqing University, Chongqing, 401147, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Yubao Cui; Dan Zeng, Email ybcui1975@hotmail.com; zengdan@cqu.edu.cn

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 E. coli 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 B. tropicalis 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.

Keywords: Blomia tropicalis, nanometer, ferritin, allergic disease, Blo t 2