Tregitope-linked Refined Allergen Vaccines for Immunotherapy in Cockroach Allergy

Abstract

Allergen-specific immunotherapy (AIT) facilitates long-term resolution of allergic morbidity resulting in reduced drug use and increased refractoriness to new sensitization. AIT effectiveness has been demonstrated in seasonal and perennial allergies, and insect stings. However, data and studies in AIT relative to cockroach (CR) allergy are relatively scarce. In this study, mice allergic to American CR (Periplaneta americana) were treated with a liposome (L)-entrapped vaccine made of mouse Tregitope289-Per a 9 of the CR, Tregitope167-Per a 9, or Per a 9 alone - or placebo. Allergic mice that received an individual vaccine intranasally had reduced Th2 response, reduced lung inflammation, and reduced respiratory tissue remodeling. However, only L-Tregitope289-Per a 9 and L-Tregitope167-Per a 9 induced expression of immunosuppressive cytokine genes (IL-10, TGF-β, and IL-35 for L-Tregitope289-Per a 9, and IL-10 and TGF-β for L-Tregitope167-Per a 9) and increment of idoleamine-2,3-dioxygenase 1 (IDO1), indicating that these vaccines caused allergic disease suppression and reversal of respiratory tissue remodeling via generation of regulatory lymphocytes. Liposome entrapped-recombinant Per a 9 (L-Per a 9) did not cause upregulation of immunosuppressive cytokine genes and IDO1 increment; rather, L-Per a 9 induced high expression of IFN-γ in lungs of treated mice, which resulted in mitigation of allergic manifestations. This study provides compelling evidence that both liposome-entrapped vaccines made of single refined major allergen alone and single refined major allergen linked with Tregitopes are effective for reducing allergen-mediated respiratory tissue inflammation and remodeling, but through different mechanisms.

Figure 1

Indirect ELISA for measuring specific IgE, IgG1 and IgG2a in sera of CRE-allergenized mice in comparison to sham and normal mice. (A) IgE, (B) IgG1, and (C) IgG2a.

Figure 2

Histological appearances and grades of lung sections of mice after staining with hematoxylin and Eosin (H & E) dyes. (A–D) Histologic grades 0–3, respectively. (E) Comparative average histological grades of normal, sham and allergenized mice. The allergenized mice had significantly higher average lung histopathological grade than the normal and sham mice (p < 0.001). The average histologic grades of normal and sham mice were not different (p > 0.05).

Figure 3

Histological appearances and grades of lung sections of mice after staining with Periodic Acid-Schiff (PAS). (A–D) Grades 0–3, respectively, of goblet cells (stained pink/red; black arrow heads) in the lung epithelia. (E) Comparative average goblet cell grades of normal, sham and allergenized mice. The allergenized mice had significantly higher average goblet cell grade than the normal and sham mice (p < 0.001). The average goblet cell grades of normal and sham mice were not different (p > 0.05).

Figure 4

Histological appearances and grades of lung sections of mice after staining with Mannson’s Trichrome (TRI). (A–D) Grades 0–3, respectively, of collagen (stained blue; black arrow heads) in the bronchiolar subepithelia. (E) Comparative average collagen grades of normal, sham and allergenized mice. The allergenized mice had significantly higher average collagen grade than the normal and sham mice (p < 0.001). The average collagen grades of normal and sham mice were not different (p > 0.05). Black arrows indicate smooth muscle cells.

Figure 5

Expressions of cytokine genes in lungs of normal, sham and CRE-allergenized mice. Allergenized mice had up-regulations of Th2 cytokine genes including IL-4, IL-5 and Il-13, IL-12a, IL-12b, IL-17a, TNF-α and IFN-γ compared to normal and sham mice. Expressions of TGF-β, IL-10 and IL-35 of all mouse groups were not different (p > 0.05).

Figure 6

Percentages of CD3⁺CD4⁺CD25^hiFoxP3⁺CD45RA⁻ T cells (Tregs) in the mouse PBMCs after exposure to: (A) medium alone (negative control), (B) tetanus toxoid which served as positive control, (C) T289, (D) T167, (E) T289-Per a 9, (F) T167-Per a 9, and (G) Per a 9 alone, for 24 hours. (H) Bar graphs for statistical comparison of the percentages of Tregs in PBMCs after different treatments.

Figure 7

Lung histopathological grades of the CR-allergic mice after immunotherapy with the L-T289-Per a 9, L-T167-Per a 9 and L-Per a 9 vaccines compared to placebo (L-P). (A) Grades of inflammatory cells that infiltrated into peribronchiolar areas, (B) Grades of goblet cells in the bronchiolar epithelia, and (C) Grades of collagen deposition around the bronchioles.

Figure 8

Cytokine expression profiles of the CRE-allergic mice treated with 8 doses of the L-T289-Per a 9, L-T167-Per a 9 and L-Per a 9 vaccines compared to placebo (L-P).

Figure 9

Levels of idoleamine-2,3-dioxygenase (IDO1) in lung tissues of allergic mice treated with T167-Per a 9, T289-Per a 9, and Per a 9 in comparison to the placebo mice. Increment of the IDO was found only in the tissues of the mouse groups treated with Tregitope-allergen fusion proteins. *Indicate significant difference (p < 0.05).