Fusion protein of TLR5-ligand and allergen potentiates activation and IL-10 secretion in murine myeloid DC

Toll-like receptor ligands are immune-modulatory components linking innate and adaptive immune responses and are considered to be promising vaccine components. Objective of this study was to investigate the adjuvant activity of Listeria monocytogenesis-derived TLR5-ligand flagellin A (flaA) genetically fused to ovalbumin (Ova, major chicken white egg allergen) in a murine in vitro system. Recombinant flaA, rOva, and a fusion protein of rflaA and rOva (rflaA:Ova) were over-expressed in Escherchia coli and purified by FPLC. LPS depletion was confirmed by LAL test. TLR5-binding was evaluated by human and murine TLR5-transgenic HEK 293 cells. The immune-modulatory effect of rflaA:Ova and rflaA:Ova modified by reduction and alkylation on purified BALB/c bone marrow-derived myeloid (mDC) and plasmacytoid dendritic cells (pDC) was investigated by flow cytometry and intracellular cytokine staining (ICS). Dose-dependent IL-8 secretion from transgenic HEK 293 cells confirmed binding of rflaA and rflaA:Ova molecules to human and murine TLR5. Recombinant flaA showed similar biological reactivity to TLR5-ligand fliC derived from Salmonella typhimurium applied as positive control. Compared to rflaA, both rflaA:Ova preparations induced higher expression of maturation markers (CD40, CD69, CD80, and CD86) on mDC, whereas only CD69 and CD40 were upregulated on pDC. Moreover, IL-6 and IL-10 production by mDC was enhanced upon stimulation with rflaA:Ova constructs in comparison to an equimolar mixture of both proteins whereas pDC did not show secretion of the investigated cytokines. Any immunological effects of LPS can be excluded by depletion of endotoxins and the lack of IL-10 production upon proteinase K digestion of rflaA:Ova. In summary, the rflaA:Ova fusion proteins showed an enhanced immune modulating capacity in comparison to rflaA or the mixture of rflaA and antigen. Since the rflaA:Ova fusion proteins induce strong IL-10 induction they are considered as potential vaccine candidates to improve allergen-specific immunotherapy