α-A26-39 rA26-39 rB ~ 270 kDa rB15-24 rA α-B ~ 308 kDa Supp. Figure 1: Toxins produced by C. difficilestrain 630. Cell-free supernatants from stationary phase cultures of strain 630 were probed with antibodies to A26-39 and B Control lanes are the purified rA26-39 and rB15-24 proteins.

TcdA TcdB TcdA TcdB TcdA TcdB TcdA TcdB TcdA TcdB Supp. Fig. 2: Homology between the cell binding domains of toxin A (TcdA ) and toxin B (TcdB ). Identical residues (30%) are shown.

116 α- CotC (1/3000) PY PY α- CotB (1/4000) CotC-A CotC CotB-A CotB-B CotB Supp. Figure 3: Analysis of CotC and CotB recombinant proteins in PP108 (CotB-A CotC-A )and PP142 (CotB-B CotC-A ). Panel A shows western blots of spore coat proteins extracted from PY79, PP108 and PP142 spores probed with antiserum to CotC (mouse polyclonal, 1/3000 dilution). The 12 kDaCotC band and the 49 kDa CotC-A bands are indicated. Panel B shows blots of coat proteins extracted from PY79, PP108 and PP142 probed with anti-CotB (mouse polyclonal, 1/4000). CotB runs at 59 kDa, recombinant CotB-A at 69 kDa and CotB-B at 60 kDa. For PP108 and PP142 the 59kDa species corresponds to CotB encoded by the endogenous cotB gene in these partial diploids where the recombinant genes are inserted in trans on the chromosome. AB

AB Supp. Figure 4. IgG1 responses in mice. IgG1 responses specific to A26-39 (panel A) and B15-24 (panel B) in mice dosed orally with PP108 ( ) and PP142 ( ) spores were determined by ELISA. Control groups were, naïve (), mice dosed with non-recombinant PY79 spores ( ) and mice dosed with a mixture of the rA26-39 and rB15-24 proteins ( ).

AB Supp. Figure 5. IgG2a responses in mice. IgG2a responses specific to A26-39 (panel A) and B15-24 (panel B) in mice dosed orally with PP108 ( ) and PP142 ( ) spores were determined by ELISA. Control groups were, naïve (), mice dosed with non-recombinant PY79 spores ( ) and mice dosed with a mixture of the the rA26-39 and rB15-24 proteins ( )

Supp. Figure 6: Isotype Ratios. The ratio of IgG1 to IgG2a antibodies obtained in serum obtained from mice dosed orogastrically with a protein mixture (rA rB15-24) or with PP108 or PP142 spores is shown. IgG values come from Supp. Figures 3 and 4. Increasing ratios indicate a Th1 bias.

Supp. Figure 7: Anti-spore Responses. IgG titres determined by indirect ELISA in mice. Plates were coated with PY79 spores (Duc et al Intracellular fate and immunogenicity of B. subtilisspores. Vaccine 22: ). The end-point IgGtiter was calculated as the dilution of serum producing the same optical density as 1/40 dilution of a pooled preimmune serum.

Toxin 630 HT29 Serum Feces PP142 Naïve PP108 Samples + Toxin VERO Serum Feces No Toxin Supp. Figure 8a: Neutralisation of cytotoxicity. HT29 and VERO cells were cultured as monolayers and their morphological phenotype examined after 24-48h of incubation. In parallel, partially purified toxin supernatants from C. difficile 630 (A + B + ) cultures were pre-neutralized (37 0 C, 1h) with antibodies from serum and fecal (pooled) diluted samples respectively obtained from immunized mice or naïve mice (1/10 dilution for serum samples and 1/50 [w:v] for feces) as shown in Figure 2 and 3. Naïve serum IgG or fecalsIgA being unable to neutralize toxins A and B exhibits the characteristic rounded cell morphology associated with cytotoxicity.

PP142 Naïve PP108 Samples + ToxinNo Toxin Toxin A (HT29) Feces Toxin B (VERO) Serum Feces Serum Supp. Figure 8b: Neutralisation of cytotoxicity. HT29 and VERO cells were cultured as monolayers and their morphological phenotype examined after 24-48h of incubation. In parallel, purified toxin A and toxin B from C. difficile 630 (A + B + ) cultures were pre-neutralized (37 0 C, 1h) with antibodies from serum and fecal (pooled) diluted samples respectively obtained from immunized mice or naïve mice (1/10 dilution for serum samples and 1/50 [w:v] for feces) as shown in Figure 2 and 3. Naïve serum IgG or fecalsIgA being unable to neutralize toxins A and B exhibits the characteristic rounded cell morphology associated with cytotoxicity.