Allergy Asthma Immunol Res.  2019 Mar;11(2):254-266. 10.4168/aair.2019.11.2.254.

A Fusion Protein of Derp2 Allergen and Flagellin Suppresses Experimental Allergic Asthma

• Tan W
• Zheng JH
• Duong TM
• Koh YI
• Lee SE
• Rhee JH

• KMID: 2431857
• DOI: http://doi.org/10.4168/aair.2019.11.2.254

Abstract

PURPOSE
The house dust mite (HDM) is one of the most important sources of indoor allergens and a significant cause of allergic rhinitis and allergic asthma. Our previous studies demonstrated that Vibrio vulnificus flagellin B (FlaB) plus allergen as a co-treatment mixture improved lung function and inhibited eosinophilic airway inflammation through the Toll-like receptor 5 signaling pathway in an ovalbumin (OVA)- or HDM-induced mouse asthma model. In the present study, we fused the major mite allergen Derp2 to FlaB and compared the therapeutic effects of the Derp2-FlaB fusion protein with those of a mixture of Derp2 and FlaB in a Derp2-induced mouse asthma model.
METHODS
BALB/c mice sensitized with Derp2 + HDM were treated with Derp2, a Derp2 plus FlaB (Derp2 + FlaB) mixture, or the Derp2-FlaB fusion protein 3 times at 1-week intervals. Seven days after the final treatment, the mice were challenged intranasally with Derp2, and airway responses and Derp2-specific immune responses were evaluated.
RESULTS
The Derp2-FlaB fusion protein was significantly more efficacious in reducing airway hyperresponsiveness, lung eosinophil infiltration, and Derp2-specific IgE than the Derp2 + FlaB mixture.
CONCLUSIONS
The Derp2-FlaB fusion protein showed a strong anti-asthma immunomodulatory capacity, leading to the prevention of airway inflammatory responses in a murine disease model through the inhibition of Th2 responses. These findings suggest that the Derp2-FlaB fusion protein would be a promising vaccine candidate for HDM-mediated allergic asthma therapy.

Keyword

Asthma; Derp2; FlaB flagellin; fusion protein; Toll-like receptor 5

MeSH Terms

Allergens
Animals
Asthma*
Eosinophils
Flagellin*
Immunoglobulin E
Inflammation
Lung
Mice
Mites
Ovalbumin
Pyroglyphidae
Rhinitis, Allergic
Therapeutic Uses
Toll-Like Receptor 5
Vibrio vulnificus
Allergens
Flagellin
Immunoglobulin E
Ovalbumin
Therapeutic Uses
Toll-Like Receptor 5

Figure

• 

  Fig. 1 Recombinant protein construction and analysis. (A) Map of expression vectors used to manufacture the Derp2, Derp2-FlaB and FlaB-Derp2 recombinant proteins. DNA fragments of derp2, flaB, the T7 promoter region, kanamycin resistance gene (Km), and cloning restriction enzyme sites are shown. (B) SDS-PAGE and Western blot analysis of the bacterially expressed and purified Derp2, Derp2-FlaB and FlaB-Derp2 recombinant proteins. The Derp2-FlaB and FlaB-Derp2 proteins were probed with a FlaB antibody. (C) TLR5-mediated NF-κB luciferase activity assay with FlaB, Derp2-FlaB, or FlaB-Derp2 fusion protein. Analysis was carried out using a dual-luciferase reporter assay system, and results were normalized to pCMV-β-galactosidase as a control. All values represent the mean plus the standard error of at least 3 independent experiments. FlaB, flagellin B; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TLR5, Toll-like receptor 5; FlaB, flagellin B. *P < 0.001 compared with FlaB-only.

• 

  Fig. 2 Experimental protocols. BALB/c mice were sensitized by intraperitoneal injections of Derp2 + HDM in alum on days 0, 7 and 14. On days 4, 11 and 18, the mice were treated intranasally with Derp2, Derp2 + FlaB, Derp2-FlaB fusion protein or FlaB under anesthesia. The mice were challenged daily by the intranasal administration of Derp2 on days 25 to 31. The control animals were treated and challenged with PBS. Parameters of allergic responses were evaluated 24 hours after the final Derp2 challenge. HDM, house dust mite; FlaB, flagellin B; PBS, phosphate-buffered saline; AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage.

• 

  Fig. 3 Assessment of airway hyperresponsiveness following allergen sensitization and challenge. Airway responsiveness to methacholine was determined in the PBS, Derp2, Derp2 + FlaB and Derp2-FlaB groups (n = 5 for each). The data are expressed as the mean ± SEM. PBS, phosphate-buffered saline; FlaB, flagellin B; SEM, standard error of mean; ns, not significant. *P < 0.05, †P < 0.01.

• 

  Fig. 4 Histological evaluation of lung tissues. Lung tissues were fixed with 10% formalin. Lung sections were stained with H&E and observed by optical microscopy (× 200). Representative images were obtained from the histology sections from the (A) PBS, (B) Derp2, (C) Derp2 + FlaB, (D) Derp2-FlaB and (E) FlaB groups. H&E, hematoxylin and eosin; PBS, phosphate-buffered saline; FlaB, flagellin B.

• 

  Fig. 5 Derp2-specific serum IgE levels in tested mice determined by ELISA. The data are expressed as the mean ± SEM. IgE, immunoglobulin E; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of mean; FlaB, flagellin B; PBS, phosphate-buffered saline; ns, not significant. *P < 0.01, †P < 0.001.

• 

  Fig. 6 Inflammatory cell and cytokine analysis of the BAL fluid. (A) BAL cells. BAL was carried out 24 hours after methacholine challenge in the mice. Cytokines IL-4 (B), IL-5 (C) and IL-13 (D) in BAL fluid were determined by ELISA. The data are expressed as the mean ± SEM. BAL, bronchoalveolar lavage; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of mean; FlaB, flagellin B; PBS, phosphate-buffered saline; ns, not significant. *P < 0.05, †P < 0.01, ‡P < 0.001.

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