A Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria

Generation of hypoallergenic neoglycoconjugates for dendritic cell targeted vaccination: a novel tool for specific immunotherapy

Esther E. Weinbergera, Martin Himlya, Julia Myschikb, Michael Hausera, Friedrich Altmannc, Almedina Isakovica, Sandra Scheiblhofera, Josef Thalhamera*, Richard Weissa a Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria b Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians- University Munich, 81377 Munich, Germany c Department of Biochemistry, University of Natural Resources and Life Sciences, 1190 Vienna, Austria

* To whom correspondence should be addressed: Josef Thalhamer, Phone: +43 662 8044 5737, Fax: 5751, email: [email protected] Supplementary Fig. S1. A) Proteolytic activity of neoglycocomplexes. SDS-PAGE and coomassie stain of a rat IgG digest using increasing dosages (1-3) of papain (Pap) activated with (+) or without (-) L-cysteine (upper graph), or MN-Pap, MD-Pap and Dex40-Pap (lower graph). B) Aldehyde content of increasingly oxidized mannans (MN1-MN4) and unmodified mannan (MN0). Supplementary Fig. S2. MN-Ova purification by SEC using a Superdex 75 GL column. A) High molecular weight complexes that are contained in fractions 4 – 10 were pooled (see also supplementary Fig.S3). B) Ova chromatogram as control. Supplementary Fig. S3. SDS-PAGE of fraction samples 3-11 of MN-Ova after SEC using a Superdex 75 GL column. High molecular weight complexes that are contained in fractions 4 – 10 of the SEC run presented in supplementary Fig.S2A were pooled. M= molecular weight size marker.

Supplementary Fig. S4. Uptake of pHrodo-labeled neoglycomplexes vs. unmodified allergens by APCs. BMDCs were incubated with different doses of antigen (x-axis) and uptake was evaluated 10, 30 and 60 minutes after addition via FACS analysis (A-C). D) depicts the time-dependent uptake of antigen at the highest dose tested (30µg/mL). Moreover, expression levels of MHC-II, CD40 and CD86 were analyzed after 60 minutes (E). Fluorescence microscopy confirmed in vitro antigen ingestion (here MN-Ova at 20µg/mL) by BMDCs (F). In vivo application of MN-Ova (10µg) into the mouse pinna revealed antigen-positive cells in the draining cervical lymph node 30 minutes after application (G). pHrodo-labeled antigen in lysosomes is shown in red. Nuclei were visualized with Hoechst dye (shown in cyan). Original magnification 60x (F&G).

Supplementary Fig. S5. Sensitization of murine basophils by Pap activated without (-) or with L- cysteine (+), Pap(E64), Ova, MN-Pap, MN-Ova, MD-Pap, Dex-Pap immunization was determined by FACS analysis. A&B) Gating strategy for murine basophils obtained from whole blood of immunized mice characterized by their CD45interCD49b+CD200R3+ phenotype. C&D) To assess basophil activation, whole blood was ex vivo stimulated with Ova or Pap(E64), respectively. Data is displayed as x-fold induction of up-regulated CD200R of antigen-stimulated vs. un-stimulated basophils. Anti- IgE was used as positive control (data not shown). Data are shown as individual BALB/c mice (n=5). * P < 0.05; ** P < 0.01.