Stefan Schülke

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.

Epicutaneous immune modulation with Bet v 1 plus R848 suppresses allergic asthma in a murine model

Combining allergen(s) with an adjuvant is a strategy to improve the efficacy and safety of allergen‐specific immunotherapy. Here, we aimed at investigating the adjuvant effects of polyadenylic–polyuridylic acid (poly(A:U)), a TLR3 agonist, and R848 (resiquimod), a TLR7 agonist, in epicutaneous immunotherapy with Bet v 1, the major birch pollen allergen, to intervene in birch pollen allergy.

MPLA shows attenuated pro‐inflammatory properties and diminished capacity to activate mast cells in comparison with LPS

Monophosphoryl lipid A (MPLA), a nontoxic TLR4 ligand derived from lipopolysaccharide (LPS), is used clinically as an adjuvant in cancer, hepatitis, and malaria vaccines and in allergen‐specific immunotherapy. Nevertheless, its cell‐activating effects have not been analyzed in a comprehensive direct comparison including a wide range of different immune cells. Therefore, the objective of this study was the side‐by‐side comparison of the immune‐modulating properties of MPLA and LPS on different immune cells.

Molecular cloning of plane pollen allergen Pla a 3 and its utility as diagnostic marker for peach associated plane pollen allergy.

Non‐specific lipid transfer proteins (nsLTP) are considered to provoke allergic symptoms to plane tree pollen, which are frequently associated with peach allergy.

Prevention of Intestinal Allergy in Mice by rflaA:Ova Is Associated with Enforced Antigen Processing and TLR5-Dependent IL-10 Secretion by mDC

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.

A Fusion Protein Consisting of the Vaccine Adjuvant Monophosphoryl Lipid A and the Allergen Ovalbumin Boosts Allergen-Specific Th1, Th2, and Th17 Responses In Vitro

Background. The detoxified TLR4-ligand Monophosphoryl Lipid A (MPLA) is the first approved TLR-agonist used as adjuvant in licensed vaccines but has not yet been explored as part of conjugated vaccines. Objective. To investigate the immune-modulating properties of a fusion protein consisting of MPLA and Ovalbumin (MPLA : Ova). Results. MPLA and Ova were chemically coupled by stable carbamate linkage. MPLA : Ova was highly pure without detectable product-related impurities by either noncoupled MPLA or Ova. Light scattering analysis revealed MPLA : Ova to be aggregated. Stimulation of mDC and mDC : DO11.10 CD4+ TC cocultures showed a stronger activation of both mDC and Ova-specific DO11.10 CD4+ TC by MPLA : Ova compared to the mixture of both components. MPLA : Ova induced both strong proinflammatory (IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-10) cytokine responses from mDCs while also boosting allergen-specific Th1, Th2, and Th17 cytokine secretion. Conclusion. Conjugation of MPLA and antigen enhanced the immune response compared to the mixture of both components. Due to the nonbiased boost of Ova-specific Th2 and Th17 responses while also inducing Th1 responses, this fusion protein may not be a suitable vaccine candidate for allergy treatment but may hold potential for the treatment of other diseases that require a strong stimulation of the hosts immune system (e.g., cancer).

Targeting of Immune Cells by Dual TLR2/7 Ligands Suppresses Features of Allergic Th2 Immune Responses in Mice

Background TLR ligands can promote Th1-biased immune responses, mimicking potent stimuli of viruses and bacteria. Aim To investigate the adjuvant properties of dual TLR2/7 ligands compared to those of the mixture of both single ligands. Methods Dual TLR2/7 ligands: CL401, CL413, and CL531, including CL264 (TLR7-ligand) and Pam2CysK4 (TLR2-ligand), were used. Immune-modulatory capacity of the dual ligands with the individual ligands alone or as a mixture in mouse BMmDCs, BMmDC:TC cocultures, or BMCMCs was compared and assessed in naïve mice and in a mouse model of OVA-induced intestinal allergy. Results CL413 and CL531 induced BMmDC-derived IL-10 secretion, suppressed rOVA-induced IL-5 secretion from OVA-specific DO11.10 CD4+ TCs, and induced proinflammatory cytokine secretion in vivo. In contrast, CL401 induced considerably less IL-10 secretion and led to IL-17A production in BMmDC:TC cocultures, but not BMCMC IL-6 secretion, or IL-6 or TNF-α production in vivo. No immune-modulating effects were observed with single ligands. All dual TLR2/7 ligands suppressed DNP-induced IgE-and-Ag-specific mast cell degranulation. Compared to vaccination with OVA, vaccination with the mixture CL531 and OVA, significantly suppressed OVA-specific IgE production in the intestinal allergy model. Conclusions Based on beneficial immune-modulating properties, CL413 and CL531 may have utility as potential adjuvants for allergy treatment.

Modular MLV-VLPs co-displaying ovalbumin peptides and GM-CSF effectively induce expansion of CD11b+ APC and antigen-specific T cell responses in vitro

Graphical abstract Figure. No caption available. HighlightsVLPs can be used as modular vaccines co‐displaying antigen (OVA) and adjuvant (GM‐CSF).GM‐CSF displayed on VLPs mediates expansion and proliferation of CD11b+ cells.Compared to a non‐modular mixture, modular VLPs increase specific T cell responses. &NA; The development of novel vaccination strategies is a persistent challenge to provide effective prophylactic treatments to encounter viral infections. In general, the physical conjugation of selected vaccine components, e.g. antigen and adjuvant, has been shown to enhance the immunogenicity and hence, can increase effectiveness of the vaccine. In our proof‐of‐concept study, we generated non‐infectious, replication deficient Murine Leukemia Virus (MLV)‐derived virus‐like particles (VLPs) that physically link antigen and adjuvant in a modular fashion by co‐displaying them on their surface. For this purpose, we selected the immunodominant peptides of the model antigen ovalbumin (OVA) and the cytokine granulocyte macrophage‐colony stimulating factor (GM‐CSF) as non‐classical adjuvant. Our results show that murine GM‐CSF displayed on MLV‐VLPs mediates expansion and proliferation of CD11b+ cells within murine bone marrow and total spleen cells. Moreover, we show increased immunogenicity of modular VLPs co‐displaying OVA peptides and GM‐CSF by their elevated capacity to induce OVA‐specific T cell‐activation and ‐proliferation within OT‐I and OT‐II splenocyte cultures. These enhanced effects were not achieved by using an equimolar mixture of VLPs displaying either OVA or GM‐CSF. Taken together, OVA and GM‐CSF co‐displaying MLV‐VLPs are able to target and expand antigen presenting cells which in turn results in enhanced antigen‐specific T cell activation and proliferation in vitro. These data suggest MLV‐VLPs to be an attractive platform to flexibly combine antigen and adjuvant for novel modular vaccination approaches.

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

Dendritic cells (DCs) are gatekeepers of the immune system that control induction and polarization of primary, antigen-specific immune responses. Depending on their maturation/activation status, the molecules expressed on their surface, and the cytokines produced DCs have been shown to either elicit immune responses through activation of effector T cells or induce tolerance through induction of either T cell anergy, regulatory T cells, or production of regulatory cytokines. Among the cytokines produced by tolerogenic DCs, interleukin 10 (IL-10) is a key regulatory cytokine limiting und ultimately terminating excessive T-cell responses to microbial pathogens to prevent chronic inflammation and tissue damage. Because of their important role in preventing autoimmune diseases, transplant rejection, allergic reactions, or in controlling chronic inflammation DCs have become an interesting tool to modulate antigen-specific immune responses. For the treatment of allergic inflammation, the aim is to downregulate allergen-specific T helper 2 (Th2) responses and the associated clinical symptoms [allergen-driven Th2 activation, Th2-driven immunoglobulin E (IgE) production, IgE-mediated mast cell and basophil activation, allergic inflammation]. Here, combining the presentation of allergens by DCs with a pro-tolerogenic, IL-10-producing phenotype is of special interest to modulate allergen-specific immune responses in the treatment of allergic diseases. This review discusses the reported strategies to induce DC-derived IL-10 secretion for the suppression of allergen-specific Th2-responses with a focus on IL-10 treatment, IL-10 transduction, and the usage of both whole bacteria and bacteria-derived components. Interestingly, while IL-10-producing DCs induced either by IL-10 treatment or IL-10 transduction are arrested in an immature/semi-mature state, treatment of DCs with live or killed bacteria as well as isolated bacterial components results in the induction of both anti-inflammatory IL-10 and pro-inflammatory, Th1-promoting IL-12 secretion often paralleled by an enhanced expression of co-stimulatory molecules on the stimulated DCs. By the secretion of DC-derived exosomes or CC-chemokine ligand 18, as well as the expression of inhibitory molecules like cytotoxic T lymphocyte-associated antigen 4, TNF receptor superfamily member 4, Ig-like transcript-22/cluster of differentiation 85, or programmed death-1, IL-10-producing DCs have been repeatedly shown to suppress antigen-specific Th2-responses. Therefore, DC-based vaccination approaches hold great potential to improve the treatment of allergic diseases.

Monophosphoryl Lipid a as an adjuvant for immune therapy? A detailed in vitro comparison to LPS

Monophosphoryl lipid A (MPL) is a non-toxic TLR4 ligand, derived from Salmonella minnesota R595 (Re) lipopolysaccharide (LPS) by chemical modification. It is clinically used as an adjuvant for cancer treatment (Fendrix®, Ceravix®) and allergen specific immunotherapy (Pollinex® Quattro, ORALVAC®). Nevertheless, reports on the mechanism of adjuvant activity are limited. The aim of this study was to compare the immune modulating capacities of MPL and LPS in vitro. In both human and murine lung epithelial cell lines (LA-4, A549) LPS induced a higher CCL2 secretion than MPL. In murine BM-derived myeloid dendritic cells (mDC), LPS as well as MPL stimulation resulted in the same pattern of cytokine secretion (IL-1β, IL-6, IL-10 and TNF-α). At high concentrations of MPL, IL-1β secretion was 4-fold higher compared to LPS, whereas LPS stimulation resulted in higher secretion of IL-6, IL-10 and TNF-α, respectively. Moreover, mDC stimulation with both adjuvants resulted in a pronounced cell activation pattern characterized by CD40 and CD69 upregulation, at which LPS proved to be more potent than MPL (thresholds for mDC activation: MPL: 100 ng/ml, LPS: 1 ng/ml). In MyD88-/- and Trif-/- mDC, MPL-induced cytokine secretion was absent in MyD88- but only reduced in Trif-deficient mDC. LPS induced cytokine secretion was mostly unchanged in Trif-/- mDC. Furthermore, the co-administration of MPL and Ova resulted in enhanced IFN-γ and IL-5 secretion from OVA-specific DO11.10 CD4+ T cells co-cultured with BALB/c mDC which was not observed for LPS controls. In line with this result, stimulation with a covalent fusion protein of MPL and Ova (MPL:Ova) resulted in enhanced cytokine secretion from both mDC (IL-1β, IL-6, TNF, IL-10, IL-12) and CD4 T cells (IL-5, IL-13, IL-2, IFN-γ, IL-17) compared to equimolar concentrations of MPL and Ova provided individually or as a mixture. Interestingly, Ova induced IL-9 secretion from CD4+ T cells was dose-dependently repressed when fused to MPL. In summary, using in vitro assay systems we observed similar but attenuated immune responses induced by MPL in comparison to LPS. MPL applied together with allergen (either mixed or covalently fused) on CD4+ T cells boosted allergen-specific TH1-, TH2-, and TH17-adaptive responses. Although considered safe in humans, further studies should critically assess the adjuvant capacity of MPL in order to evaluate potential non-desired immunological effects.