Fusion Proteins of Flagellin and the Major Birch Pollen Allergen Bet v 1 show Enhanced Immunogenicity, Reduced Allergenicity and Intrinsic Adjuvanticity

Publication date: Available online 26 April 2017
Source:Journal of Allergy and Clinical Immunology
Author(s): Claudia Kitzmüller, Julia Kalser, Sonja Mutschlechner, Michael Hauser, Gerhard J. Zlabinger, Fatima Ferreira, Barbara Bohle
BackgroundRecombinant fusion proteins of flagellin and antigens have been demonstrated to induce strong innate and adaptive immune responses. Such fusion proteins may enhance the efficacy of allergen-specific immunotherapy (AIT).ObjectiveTo characterize different fusion proteins of flagellin and the major birch pollen allergen Bet v 1 for suitability as allergy vaccines.MethodsA truncated version of flagellin (NtCFlg) was genetically fused to the N- or C-terminus of Bet v 1. TLR5-binding was assessed with HEK293 cells expressing TLR5. Up-regulation of CD40, CD80, CD83, and CD86 on monocyte-derived dendritic cells (mdDC) from allergic patients was analyzed by flow cytometry. The T cell-stimulatory capacity of the fusion proteins was assessed with naïve and Bet v 1-specific T cells. IgE-binding was tested in inhibition ELISA and basophil activation tests (BAT). Mice were immunized with the fusion proteins in the absence and presence of aluminium hydroxide (alum). Cellular and antibody responses were monitored. Murine antibodies were tested for blocking capacity in BAT.ResultsBoth fusion proteins matured mdDC via TLR5. Compared to Bet v 1 the fusion proteins showed stronger T cell-stimulatory and reduced IgE-binding capacity and induced murine Bet v 1-specific antibodies in the absence of alum. However, only antibodies induced by immunization with NtCFlg fused to the C-terminus of Bet v 1 inhibited the binding of patients´ IgE antibodies to Bet v 1.ConclusionBet v 1-flagellin fusion proteins show enhanced immunogenicity, reduced allergenicity and intrinsic adjuvanticity and thus, represent promising vaccines for birch pollen AIT. However, the sequential order of allergen and adjuvant within a fusion protein determines its immunological characteristics.

Teaser

Bet v 1-flagellin fusion proteins display several advantages over wild-type Bet v 1, namely reduced allergenicity, immunomodulatory capacity, enhanced immunogenicity and intrinsic adjuvanticity.


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