Conjugation of wildtype and hypoallergenic mugwort allergen Art v 1 to flagellin induces IL-10-DC and suppresses allergen-specific TH2-responses in vivo

Abstract

Allergies to weed pollen including members of the Compositae family, such as mugwort, ragweed, and feverfew are spreading worldwide. To efficiently treat these newly arising allergies, allergen specific immunotherapy needs to be improved. Therefore, we generated novel vaccine candidates consisting of the TLR5-ligand Flagellin A from Listeria and the major mugwort allergen Art v 1 including either the wild type Art v 1 sequence (rFlaA:Artv1) or a hypoallergenic variant (rFlaA:Artv1hyp) with reduced IgE-binding capacity. Immune modulating capacity of these constructs and respective controls was evaluated in vitro and in vivo. Incorporation of hypoallergenic Art v 1 derivative did not interfere with the resulting fusion proteins’ immune stimulatory capacity. Both rFlaA:Artv1 and rFlaA:Artv1hyp induced a prominent, mTOR-dependent, IL-10 secretion from murine dendritic cells, and suppressed allergen-specific TH2-cytokine secretion in vitro and in vivo. Both conjugates retained the capacity to induce rFlaA-specific antibody responses while efficiently inducing production of Art v 1-specific IgG1 and IgG2a antibodies in mice. Interestingly, only the suppression of TH2-cytokine secretion by rFlaA:Artv1 (but not rFlaA:Artv1hyp) was paralleled by a strong secretion of IFN-γ. In summary, we provided evidence that incorporating hypoallergens into flagellin:allergen fusion proteins is a suitable strategy to further improve these promising vaccine candidates.

Induction of Interleukin-10 Producing Dendritic Cells As a Tool to Suppress Allergen-Specific T Helper 2 Responses

“…These findings show that interestingly, the immune-modulatory cytokine secretion, and therefore the DC tolerizing capacity, of this vaccine candidate was linked to the activation of mDC metabolism. Similar results were obtained for a fusion construct consisting of FlaA and the major mugwort allergen Art v 1 ( 95 ).…”

“…When co-cultured with allergen-specific CD4 + T cells, these DCs efficiently suppressed both allergen-induced Th1 and Th2 cytokine secretion in vitro (Figure 3 B) ( 93 , 95 , 96 ). In this experimental setting, the flagellin fusion protein-mediated suppression of both Th1- and Th2-cytokine secretion was dependent on IL-10, since this effect was reversed when using either IL-10-neutralizing antibodies or IL-10-deficient mDC as APCs for the co-cultures ( 93 ).…”

“…In our own studies, we evaluated the induction of IL-10-producing mDCs using fusion proteins consisting of the recombinant TLR5-ligand flagellin A (rFlaA) from L. monocytogenes and either OVA from hen’s egg as a model allergen (rFlaA:OVA) ( 92 – 94 ), the major mugwort allergen Artemisia vulgaris allergen 1 (Art v1) (rFlaA:Artv1) ( 95 ), or the major birch pollen allergen Betula verrucosa allergen 1 (Bet v 1) (rFlaA:Betv1) ( 96 ). Such fusion proteins efficiently target TLR5 + immune cells, e.g., DCs, macrophages, and epithelial cells which take up, process, and present the fused antigen in the context of the flagellin-mediated cell activation.…”

“…Mouse bone marrow-derived mDCs stimulated with such fusion proteins were strongly activated [upregulation of CD40, CD69, CD80, CD86, programmed cell death 1 ligand 2 (B7-DC, CD273), PD-L1], displayed highly increased levels of the target receptor TLR5 on their cell surface, and secreted both pro- (IL-1β, IL-6) and anti-inflammatory (IL-10) cytokines (Figure 3 A) ( 93 , 95 , 96 ). Interestingly, the non-fused mixture of both components (flagellin and allergen) did not have a comparable effect ( 93 , 95 , 96 ).…”

Molecular approaches to allergen‐specific immunotherapy: Are we so far from clinical implementation?

“…Listeria monocytogenesis ‐derived flagellin A, a strong TLR5 ligand, was genetically fused to ovalbumin or pollen Art v 1 and Bet v 1 allergens for the efficient antigen targeting to TLR5 positive APCs . Flagellin‐allergen fusion proteins were shown to generate in vitro IL‐10‐producing tolerogenic DCs capable to suppress Th1 and Th2 cytokine responses.…”