Fig 2: Anti-Dsg1 and -Dsg3 antibodies did not result from a cross-reactivity with PpSP32.The preimmune sera as well as sample 8 (recovered at day 120) of mice immunized with rPpSP32 were either not preincubated or preincubated with increasing concentrations of rPpSP32 and then tested by ELISA to detect anti-PpSP32, -Dsg1, or -Dsg3 antibodies. *P < 0.05. Student’s t test used when compared with S8 (for IgG anti-PpSP32, P = 0.0052, P = 0.0072, and P = 0.0002 for the conditions with PpSP32 at 5 µg/mL, 10 µg/mL, and 50 µg/mL, respectively).

Fig 3: Development of anti-Dsg1 and -Dsg3 antibodies in mice immunized with PpSP32.Mice were immunized intradermally with 2 µg rPpSP32 twice weekly over 30 days and then once per week for 90 days. (A) The sera, recovered every 15 days (samples 1–8) were tested by ELISA to detect total IgG antibodies anti-PpSP32, -Dsg1, and -Dsg3. *P < 0.05. Student’s t test when compared with sample 1. For anti-Dsg1 antibodies, P = 0.009, P = 0.01, and P = 0.01 at the sixth, seventh, and eighth sample, respectively. For anti-Dsg3 antibodies, P = 0.008, P = 0.025, and P = 0.0016 at the sixth, seventh, and eighth sample, respectively. (B) Sera recovered at day 120 (sample 8) were tested by Western blot against rPpSP32, partial Dsg1 (rDsg1, MW 77 KDa), or partial Dsg3 (rDsg3, MW 79 KDa). The result is representative of 3 independent experiments. (C) Preimmune sera and sera recovered at day 120 (sample 8) were tested by immunofluorescence on monkey esophagus substrate. A representative photo is shown. Original magnification, ×40. (D) Preimmune sera and sera recovered at day 120 (sample 8) were tested by immunofluorescence on monkey esophagus substrate and mouse tail and tongue biopsies. Representative photos for H&E staining and indirect immunofluorescence (IFI) are shown. Original magnification, ×40.

Fig 4: Binding of rPpSP32 to an epidermal protein of approximately 170 kDa demonstrated by a far-Western blot.The proteins of an epidermal extract were separated by PAGE. After being denatured and renatured, the proteins were either not incubated or incubated with the recombinant protein rPpSP32. (A) The putative interaction of rPpSP32 with 1 or more epidermal proteins was revealed using a peroxidase-labeled anti-His antibody. Controls correspond to the epidermal extract or the rPpSP32 His-tagged protein alone. The figure is representative of 3 independent experiments. (B) Dsg1 and Dsg3 were visualized in the epidermal extract using monoclonal anti-Dsg1 and -Dsg3 antibodies. MW marker, RPN 800E (Amersham), was used in this experiment.

Fig 5: ELISA showing a specific and dose-dependent interaction between PpSP32 and the proteins Dsg1 and Dsg3.The ELISA plates were coated with the recombinant proteins Dsg1 and Dsg3 or other epidermal proteins; BP180, desmocollin 3 and desmoplakin and then incubated with a decreasing concentration of rPpSP32, the protein of interest or CFP32, a control protein. The plates were then incubated in the presence of human sera with anti-PpSP32 or -CFP32 antibodies. The results are representative of 3 independent experiments. Significant differences were found between the different PpSP32 concentrations for Dsg1 and Dsg3 (P = 0.006 and P = 0.008 between the condition 2 µg/mL and 0.0002 µg/mL for Dsg1 and Dsg3, respectively). For the control proteins, no significant difference was obtained while decreasing the concentration of PpSP32 (P > 0.05).