ELECTRONIC SUPPLEMENTARY MATERIAL
Next-generation active immunization approach for synucleinopathies - implications for
Parkinson’s Disease clinical trials
Markus Mandler1, Elvira Valera2, Edward Rockenstein2, Harald Weninger1, Christina Patrick2, Anthony
Adame2, Radmila Santic1, Stefanie Meindl1, Benjamin Vigl1, Oskar Smrzka1,Achim Schneeberger1, Frank
Mattner1, Eliezer Masliah2,3
AFFiRiS AG1,Vienna Biocenter, A-1030 Vienna, Austria
Departments of 2Neurosciences and 3Pathology,
University of California, San Diego, La Jolla, California 92093, USA
Note: Markus Mandler and Elvira Valera are co-first authors
Correspondence and reprint requests should be addressed to: Eliezer Masliah, M.D. University of
California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0624. Phone: 858-534-8992,
Fax: 858-534-6232, email: emasliah@ucsd.edu
SUPPLEMENTAL MATERIALS AND METHODS
Determination of T cell responses
C57BL/6J mice were immunized at biweekly intervals and sacrificed three days after the
fourth injection. Spleen and draining lymph nodes (inguinal, axillary and brachial) were excised,
and T cell responses quantified using an ELISPOT Plus kit (Mabtech, Nacka Strand, Sweden) for
IL-4 and IFN-γ according to the manufacturer's instructions. Cells were stimulated in triplicates
with 10 µg/ml of AFF peptides, 100 µg/ml of KLH (Sigma) and 3.8 µg/ml of -syn (rPeptide) or
medium (mock stimulated). The net spot number (counted spot number minus mock stimulated
spot number) and the mean for each animal and condition were calculated.
Epitope masking assay
In order to determine if the binding of AFF 1-induced antibodies would mask the LB509
epitope, brain sections from non-tg and PDGF-α-syn tg mice were pre-incubated overnight with
the purified, FITC-tagged monoclonal antibody mAb-AFF1 (1:100), and then immunostained
with LB509 (1:250). LB509 antibody binding was detected using either diaminobenzidine, or
Tyramide Signal Amplification™-Direct (Red) system (1:100, NEN Life Sciences). Sections
were analyzed with a digital B50 Olympus microscope.
Inducible Nitric Oxide Synthase immunoblot detection
Immunoblot analysis of inducible nitric oxide synthase (iNOS) levels was performed in
soluble fractions of brain homogenates, following the same procedure as described in Materials
and Methods. The primary antibody used for detecting iNOS is from Millipore (1:1000).
Incubation with the primary antibody was followed by incubation with secondary antibody
tagged with horseradish peroxidase (1:5000, Santa Cruz Biotechnology), visualization with
enhanced chemiluminescence, and analysis with a Versadoc XL imaging apparatus (BioRad).
Analysis of -actin (Sigma) levels was used as a loading control.
SUPPLEMENTAL FIGURE LEGENDS
Suppl. Fig. 1 T-cell response to immunization with AFF peptides. T-cells responses were
measured by ELISPOT analysis following vaccination of C57BL/6J mice with AFF 1 or AFF 2.
(a) Secretion of IFN-γ following splenocyte re-stimulation with PMA-Ionomycin/A23
(PMA/A23187), carrier (KLH), -syn, and the peptide moieties of AFF 1 and 2. (b) Secretion of
IL-4 following splenocyte re-stimulation with PMA-Ionomycin/A23 (PMA/A23187), carrier
(KLH), -syn and the peptide moieties of AFF 1 and 2. In both cases, results are expressed as
number of spot-forming cells (SFCs)/2.5 x 105 cells ± SEM. (c) Immunostaining of T-cells
present in the perivascular space with an anti-CD4 antibody. CD4-positive cells were observed in
positive controls (EAE), but only rare CD4-positive cells were observed in immunized animals.
BV, blood vessel. Scale bar = 10 μm
Suppl. Fig. 2 Binding of AFF 1-induced antibodies to -syn did not interfere with the binding of
the LB509 antibody. Vibratome sections of non-tg mice and PDGF--syn tg mice were stained
with LB509 alone (1:250), FITC-tagged mAb-AFF1 alone (1:100), or first pre-incubated with
FITC-tagged mAb-AFF1 and then stained with LB509. (a) Antibody binding as detected by
diaminobenzidine staining. Scale bar = 10 μm (b) Antibody binding as detected by
immunofluorescence. LB509 binding was detected by tyramide red signal amplification (red),
and mAb-AFF1 by FITC fluorescence (green). Cell nuclei were stained with DAPI (blue). Scale
bar = 5 μm
Suppl. Fig. 3 Effect of vaccination with AFF 1 on inducible nitric oxide synthase levels in
PDGF--syn tg mice. Levels of inducible nitric oxide synthase (iNOS) were measured by
immunoblot in the soluble fraction of brain protein extracts of non-tg mice and PDGF--syn tg
mice treated either with vehicle or AFF 1. (a) Immunoblot analysis of iNOS. -actin was used as
loading control. (b) Densitometric analysis of the immunoblot results. Results are expressed as
average ± SEM. (*) p<0.05