Conjugated
vaccines consisting of
flagellin and
antigen activate TLR5 and induce strong innate and adaptive immune responses. Objective of the present study was to gain further insight into the mechanisms by which
flagellin fusion
proteins mediate their immune modulating effects. In a mouse model of Ova-induced intestinal
allergy a fusion
protein of
flagellin and Ova (rflaA:Ova) was used for intranasal and intraperitoneal vaccination. Aggregation status of flaA, Ova and flaA:Ova were compared by light scattering, uptake of fluorescence labeled
proteins into mDC was analyzed, processing was investigated by microsomal digestion experiments. Mechanism of DC-activation was investigated using
proteasome and
inflammasome inhibitors. Immune responses of wildtype, IL-10(-/-), TLR5(-/-) mDCs and Ova-transgenic T cells were investigated. Mucosal and i.p.-application of rflaA:Ova were able to prevent allergic sensitization, suppress disease-related symptoms, prevent
body weight loss and reduction in food uptake. Intranasal vaccination resulted in strongest suppression of Ova-specific
IgE production. These protective effects were associated with increased aggregation of rflaA:Ova and accompanied by tenfold higher uptake rates into mDC compared to the mixture of both
proteins. Microsomal digestion showed that stimulation with rflaA:Ova resulted in faster degradation and the generation of different
peptides compared to rOva. rflaA:Ova-mediated activation of mDC could be suppressed in a dose-dependent manner by the application of both
inflammasome and
proteasome inhibitors. Using TLR5(-/-) mDC the rflaA:Ova induced
IL-10 secretion was shown to be TLR5 dependent. In co-cultures of IL-10(-/-) mDC with DO11.10 T cells the lack of rflaA:Ova-mediated
IL-10 secretion resulted in enhanced levels of both TH2 (IL-4, IL-5) and TH1 (IL-2 and IFN-y)
cytokines. In summary, mucosal vaccination with flaA:Ova showed strongest preventive effect. Stimulation with rflaA:Ova results in strong immune modulation mediated by enhanced uptake of the aggregated fusion
protein, likely resulting in a different processing by DC as well as stronger TLR5 mediated cell activation.