PLoS One. 2014 Feb 7;9(2):e87822. doi: 10.1371/journal.pone.0087822.
Prevention of intestinal allergy in mice by rflaA:Ova is associated with enforced antigen processing and TLR5-dependent IL-10 secretion by mDC
Stefan Schülke1*#, Sonja Wolfheimer1#, Gabriele Gadermaier2, Andrea Wangorsch1#, Susanne Siebeneicher3, Peter Briza2, Ingo Spreitzer4, Dirk Schiller1, Bettina Loeschner4, Satoshi Uematsu5, Bernard Ryffel6, Shizuo Akira5, Zoe Waibler7, Stefan Vieths1#, Masako Toda3#, Stephan Scheurer1#
1Division of Allergology, Paul-Ehrlich-Institut, Langen, Hessen, Germany
2Christian Doppler Laboratory for Allergy Diagnosis and Therapy, Department of Molecular Biology, University of Salzburg, Salzburg, Austria
3Junior Research Group “Experimental Allergy Models”, Paul-Ehrlich-Institut, Langen, Hessen, Germany
4Division of Microbiology, Paul-Ehrlich-Institut, Langen, Hessen, Germany
5Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Osaka Prefecture, Japan
6CNRS Department of Molecular and Experimental Immunology and Neurogenetics, Allergy and Lung Inflammation, University of Orléans, Orléans, Loiret département, France
7Junior Research Group “Novel vaccination strategies and early immune responses”, Paul-Ehrlich-Institut, Langen, Hessen, Germany
# Present address: Paul-Ehrlich-Institut, Vice President´s Research Group 1: Molecular Allergology
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.
Currently, conventional allergen immunotherapy (AIT) with allergen extracts is not convenient for patients because of multi-year treatment periods1. Moreover, for some allergies AIT is still less efficacious and can be hampered by unwanted side effects. To improve AIT, novel vaccine candidates and accompanying adjuvants that increase efficacy while decreasing unwanted adverse-effects are needed.
In this context, the discovery of TLR-ligands with their intrinsic ability to induce robust innate and subsequent adaptive immune responses was thought to hold great potential for the discovery and development of novel adjuvants.
Therefore, we developed a novel vaccine candidate for the treatment of allergic diseases by covalently combining the immune-activating TLR5-ligand Flagellin A (flaA) from Listeria monocytogenes with the model allergen Ovalbumin (Ova) in a conjugated fusion protein (rflaA:Ova).
Adjuvant:allergen conjugates have several advantages over simple non-conjugated mixtures of both components: (1) they target the conjugate to the respective immune cell by binding to specific immune receptors (here TLR5 which may mediate both immune-related signaling and protein uptake). Upon binding to the target cell they (2) deliver the conjugated allergen to the immune cell in the context of the adjuvant-mediated cell activation which may influence allergen uptake, processing and presentation2. Moreover, (3) adjuvant and allergen are simultaneously delivered to the same cell in a fixed molecular ratio, thereby preventing potentially detrimental bystander activation.
In previous studies we were able to show, that stimulation of myeloid dendritic cells (mDC) with rflaA:Ova resulted in strong cell activation, TLR5-upregulation, mDC-derived IL-10 secretion, and subsequent suppression of TH1 and TH2 cytokine secretion from Ova-specific CD4 T cells, while the non-fused mixture of rflaA + Ova had no effect on Ova-induced TC-differentiation3,4.
To further elucidate the events resulting in the observed mDC activation and the mechanisms by which flagellin fusion proteins induce such strong protective immune responses, we characterized the effects of rflaA:Ova stimulation on murine mDCs as target cells for a successful vaccination.
In this publication we were able to show, that rflaA:Ova formed high molecular aggregates in solution, which were likely caused by a spontaneous self-assembly of flagellin molecules as already described for bacterial flagella. These aggregates were associated with an approx. 10-fold stronger uptake of the fusion protein compared to both components provided alone or as a non-fused mixture. Uptake experiments with TLR5-deficient mDC revealed this stronger uptake to be mediated by both a receptor independent mechanism (e.g. phagocytosis) and additional TLR5-mediated endocytosis.
To further analyze the faith of rflaA:Ova within the APC we analyzed protein processing in vitro by microsomal digestion performed with mDC-derived microsomes. Treatment of rflaA:Ova with microsomal proteases resulted in both the faster generation of Ova-derived peptides and a broader spectrum of Ova-derived peptides generated. Therefore, the enforced internalization or flaA:Ova and a different accessibility to protease cleavage sites resulted in its faster and more efficient microsomal digestion degradation.
In a next step we wanted to clarify whether the observed strong IL-10 secretion by rflaA:Ova-stimulated mDC in vitro was both mediated by TLR5-activation and sufficient for the suppression of Ova-induced T cell differentiation. Using both TLR5 and IL-10 knock out mice, we were able to show, that the rflaA:Ova induced IL-10 secretion from mDC was both TLR5-dependent and sufficient for the suppression of both TH1 and TH2 cytokine secretion previously observed in co-culture experiments of mDC with Ova-specific DO11.10 CD4+ T cells in vitro3.
Finally, the immune modulating properties of the fusion protein were put to the test using a prophylactic vaccination approach in an experimental mouse model of Ova-induced intestinal allergy5. In this model both i.p and i.n. vaccination with the fusion protein were shown to efficiently prevent allergic sensitization, resulting in a reduction of clinical symptoms, reduced Ova-specific IgE levels and a predominant induction of Ova-specific IgG2a antibodies.
Therefore, we think that flagellin containing fusion proteins hold great potential as mucosal vaccines for the treatment of allergic diseases and suggest the following mechanism in order to explain the potent immune modulating properties of the flagellin:allergen fusion protein (Fig. 1):
In a first step the fusion protein is efficiently targeted to its target cells by binding to TLR5, recognized as a danger signal and taken up. Additionally, we observed an aggregation of the fusion protein, likely resulting in an enhanced TLR-independent uptake, since it has been shown multiple times that DC take up aggregates more efficiently than monomeric proteins. In a second step both fusion protein-induced TLR5 activation and stronger uptake resulted in an enforced antigen processing within the mDC. Together these processes result either alone or in conjunction with other yet unknown processes in a strong up-regulation of the target receptor TLR5, which in turn via binding of more fusion protein molecules to TLR5 leads in a positive feedback loop to the observed stronger mDC activation. Finally, the IL-10 secreted upon mDC activation suppressed the production of both TH1 and TH2 cytokines from Ova-specific T cells. In contrast to this the simple mixture of flagellin A and Ova induced only low dendritic cell activation and cytokine secretion which was not able to interfere with Ova-induced T cell differentiation.
This work was funded by the German Research Foundation (DFG SCHE 637/3-1).
- Justicia JL, Cardona V, Guardia P, Ojeda P, Olaguíbel JM, Vega JM, et al. Validation of the first treatment-specific questionnaire for the assessment of patient satisfaction with allergen-specific immunotherapy in allergic patients: the ESPIA questionnaire. J Allergy Clin Immunol 2013;131:1539–46.
- Khan S, Bijker MS, Weterings JJ, Tanke HJ, Adema GJ, van Hall T, et al. Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells. Journal of Biological Chemistry 2007;282:21145–59.
- Schülke S, Burggraf M, Waibler Z, Wangorsch A, Wolfheimer S, Kalinke U, et al. A fusion protein of flagellin and ovalbumin suppresses the TH2 response and prevents murine intestinal allergy. J Allergy Clin Immunol 2011;128:1340–1348.e12.
- Schülke S, Waibler Z, Mende M-S, Zoccatelli G, Vieths S, Toda M, et al. Fusion protein of TLR5-ligand and allergen potentiates activation and IL-10 secretion in murine myeloid DC. Mol Immunol 2010;48:341–50.
- Burggraf M, Nakajima-Adachi H, Hachimura S, Ilchmann A, Pemberton AD, Kiyono H, et al. Oral tolerance induction does not resolve gastrointestinal inflammation in a mouse model of food allergy. Mol Nutr Food Res 2011;55:1475–83.
Stefan Schülke, PhD
Paul-Ehrlich-Institut, Vice President´s Research Group 1: Molecular Allergology
Paul-Ehrlich-Str. 51-59, 63225 Langen, GERMANY
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