Supp.

Figure 1: Saturation of binding

Bound protein (μg) / 2×109 spores

protein (μg)

Purified recombinant proteins, TTFC (○), GST-Cpa247-370 (▲) or PA () were mixed with 2 X 109 PY79 spores (TTFC and
GST-Cpa247-370 ) or HU58 (PA) and incubated at RT for 1h. Spores were centrifuged, washed two times and coat proteins
extracted and spore-associated protein detected by Western blotting using appropriate antibodies
 Supp. Figure 2a and 2b:
GST-Cpa247-370 -specific IgG1:IgG2a ratios

3.5 2
a (Oral) b (Nasal)
1.8
3
1.6

2.5 1.4

1.2
2

Ratio IgG1/IgG2a
Ratio IgG1/IgG2a

1.5
0.8

1 0.6

0.4
0.5
0.2

0 0
P Y 79 + G S T -C pa H T 251 P Y 79+ G S T -C p a H T 25 1

D ay 20 D ay 4 0 D ay 60 D a y 20 D a y 40 D ay 6 0
 Supp. Figure 2c:
PA-specific IgG1:IgG2a ratios

IgG1/IgG2a ratio

day 21 day 42 day 63

 Supp. Figure 2d:
End-point titer TTFC-specific IgG subclasses

naive PY79 TTFC RH103 PY79 PY79 PY79

(1 µ g) + + Autoclaved
TTFC TTFC +
(5µ g) (1 µ g) TTFC
+ (1 µ g)
WASH
Supp. Fig. 2: Antigen-specific IgG1: IgG2a ratios

Serum samples taken from immunisation experiments (described in the text) were analysed for the IgG1 and
IgG2a subclasses and the relative ratios of IgG1 to IgG2a shown.

Panel A: oral immunisations with spores adsorbed with GST-Cpa247-370 ,

Panel B: nasal immunisations with spores adsorbed with GST-Cpa247-370 ,

Panel C: nasal immunisations with spores adsorbed with PA,

Panel D: Antibody titres of IgG1, IgG2a and IgG2b from mice immunised nasally with spores adsorbed with
TTFC.
 Supp. Figure 3a: TTFC-specific IFN-γ

5000

4500

4000

3500
IFN-γ (pg/ml)

3000

2500

2000

1500

1000

500

0
naive PY79 TTFC RH103 PY79 PY79 PY79
+ + AC
TTFC TTFC +
washed TTFC
 Supp. Figure 3b: PA-specific IFN-γ

IFN-γ (pg/ml)

Naïve HU58 PA HU58

+
PA
Supp. Fig. 3: Antigen-specific IFN-γ production

IFN-γ responses determined by ELISA from mice immunised with spores as indicated. Splenocytes were
extracted from sacrificed mice and re-stimulated with TTFC (Panel A) or PA (Panel B) protein (5
µ g/ml). IFN-γ was determined from supernatants after six days incubation.