SI Materials and Methods
Reagents and Abs.
Isopropyl β-D-thiogalactopyranoside, SIGMAFAST™ OPD substrate tablets, OVA
grade VI, propidium iodide and 7-Amino-actinomycin were purchased from Sigma (St
Louis, MO). Mouse IFNγ was purchased from PreproTech EC Ltd. (London, UK).
Complete, EDTA-free protease inhibitor cocktail tablets were purchased from Roche
(Meylan, France). Alexa Fluor® 647 microscale protein labeling kit was purchased from
Molecular Probes, Inc. (Eugene, OR). Abs for immunofluorescence studies included
mouse anti-histidine Ab (Euromedex, Mundolsheim, France), sera from Pt-Dd
immunized rabbits [previously described by Fender et al. (1)] and Alexa 488 anti-mouse
and Alexa 546 anti-rabbit Ab (Molecular probes). Abs for ELISA analysis included
HRP-conjugated sheep anti-mouse IgG (GE Healthcare, Orsay, France), mouse Ab
isotyping reagents (goat anti-mouse IgG1, IgG2a, IgG2b, IgG3 and IgA, SigmaAldrich) and HRP-conjugated rabbit anti-goat IgG (Sigma). FITC rat anti-mouse CD8a
(Ly-2) Ab (clone 53-6.7) was purchased from BD Pharmingen™ (BD Biosciences, San
Jose, CA) and PE H-2Kb-ovalbumin257-264 (SIINFEKL) tetramer from Immunomics
(Beckman Coulter, Fullerton, CA).
Purification of WW proteins.
To purify WW, BL21 cells expressing WW protein were lysed by sonication in binding
buffer (25 mM Tris pH 7.5, 300 mM NaCl, 10 mM imidazole and protease inhibitors).
Cleared supernatant was loaded on a nickel sepharose HisGraviTrap column (GE
Healthcare). After extensive washing with washing buffer (binding buffer containing 40
mM imidazole), WW protein was eluted with elution buffer (binding buffer containing
250 mM). WW-OVA protein was purified similarly, except the binding buffer (25 mM
bicine pH 9, 300 mM NaCl, 2 µM 2-ME, 0.1 mM EDTA, 10% glycerol), the
concentration of imidazole in the washing (10 mM) and elution (40 mM) steps and the
use of chelating sepharose fast flow (GE Healthcare). Proteins were concentrated by
ultrafiltration in Vivaspin columns or Vivapore 10/20 concentrators (Sartorious,
Aubagne, France), dialysed overnight at 4°C in PBS and stored at -80°C until used.
Immunofluorescence of WW-OVA transduced HeLa cells.
Cells were extensively washed with PBS, fixed with 4% paraformaldehyde for 10 min
and permeabilized with 0.1% saponin-PBS for 6 min. Cells were blocked for 30 min in
blocking solution (0.1% saponin, 5% non-fat dry milk in PBS), followed by overnight
incubation at 4°C with mouse anti-histidine (1/200 dilution) and rabbit anti-Pt-Dd
(1/1,000 dilution) Abs. Primary Abs were recognised with Alexa 488 and 546 Abs.
Nuclei were counter stained with Hoechst dye and slides mounted with Dako mounting
medium (Dako, Glostrup,Denmark).
Cytotoxicity assay.
Splenocytes from WW-OVA/Pt-Dd or PBS vaccinated mice were co-cultured in 24well plates with B16-OVA target cells (30,000 cells/well) previously treated with 10
ng/ml mouse IFNγ for 48 h. Co-cultures were incubated for 16 h at target:effector ratios
of 1:25, 1:50 and 1:100 in complete RPMI medium with 100 IU/ml IL-2. Cultures were
harvested and a quantitative analysis of viable target cells was performed based on the
method described by Jedema et al (2). FITC-labelled calibration beads (CaliBrite™
Beads, BD Biosciences) were used as an internal reference and excluding propidium
iodide+ non-viable cells. The percentage of specific lysis was calculated with the
formula [1- (R 1:1/R 1:A)] x 100, where R represent the ratio between number of
calibration beads/number of viable B16-OVA cells for the target:effector ratio 1:1 and
1:A (where A is 25, 50 or 100).
Determination of Ab production by ELISA assay.
Immuno 96 MaxiSorp™ plates (Thermo Fisher Scientific) were coated overnight at 4°C
with 10 µg/well of OVA diluted in coating buffer (0.1 M Na2CO3 pH 9.6). Unreacted
sites were blocked with 2% BSA in PBS for 4 h at 37°C. Following washes in PBS0.05% Tween 20, 3-fold serial dilutions of each serum sample in PBS-1% BSA, 0.05%
Tween 20 were incubated for 1 h at 37°C. After washing, bound IgG from sera was
detected with sheep anti-mouse-HRP IgG diluted 1/1,500 followed by detection with
OPD substrate. Ab isotypes were determined similarly using goat anti-mouse IgG1,
IgG2a, IgG2b, IgG3 and IgA diluted 1/500 and rabbit anti-goat HRP IgG diluted
1/2,500. Absorbances were measured at O.D.450 nm using a microplate reader. Results
are represented as blank-subtracted O.D. values from each mouse serum at dilution
1/50. Statistically significant differences between the Ab titers elicited by vaccination
were analysed with GraphPad Prism 4.0 software using the unpaired t-test with Welch´s
correction. All tests were two-tailed and a value of P< 0.05 was considered statistically
significant.
SI Reference list
1.
2.
Fender P, Boussaid A, Mezin P, & Chroboczek J (2005) Synthesis, cellular
localization, and quantification of penton-dodecahedron in serotype 3
adenovirus-infected cells. Virology 340(2):167-173.
Jedema I, van der Werff NM, Barge RM, Willemze R, & Falkenburg JH (2004)
New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells
of leukemic precursor cells within a heterogeneous target cell population. Blood
103(7):2677-2682.