Immunodominant peptides of Ag 85 A from <i>M. ulcerans</i> and <i>M. tuberculosis</i>.

*<p>Bold Amino Acids are high conserved amino acid differences (determined according to the Needleman-Wunsh criterion) and Amino Acids underlined are the non-conserved aa difference in comparison to the Ag85A peptides of <i>M. tuberculosis</i>.</p

Spleen cell IL-2 (A) and IFN-γ (B) responses to RPMI medium (grey bars), recombinant Ag85A from <i>M. ulcerans</i> (white bars) and <i>M. tuberculosis</i> (black bars) in B6 mice vaccinated with Ag85A DNA, Ag85A protein or Ag85A DNA boosted with protein in Gerbu adjuvant, 3 months after the third immunization.

<p>Cytokines levels tested on 24 h (IL-2) and 72 h (IFN-γ) culture supernatant of 4 mice tested individually/group. Data presented as means±SDs intra-assay. Statistically significant results as compared to the DNA vaccinated <i>M. ulcerans</i> or <i>M. tuberculosis</i> groups are represented in the figure by *** (P<0.001), ** (P<0.01) and * (P<0.05). The first group of comparison take all of the vaccinated mice compared to the DNA vaccinated mice in response to recombinant Ag85A from <i>M. ulcerans</i>. The second group of comparison take all of the vaccinated mice compared to the DNA vaccinated mice in response to recombinant Ag85A from <i>M. tuberculosis.</i></p

Spleen cell IL-2 (A) and IFN-γ (B) responses to whole Ag85A-<i>M. tuberculosis</i> and its synthetic peptides in a pool of six B6 mice vaccinated, at three weeks interval with Ag85A-DNA <i>M. ulcerans</i> (white bars) or Ag85A-DNA <i>M. tuberculosis</i> (black bars).

<p>Spleen cell IL-2 (A) and IFN-γ (B) responses to whole Ag85A-<i>M. tuberculosis</i> and its synthetic peptides in a pool of six B6 mice vaccinated, at three weeks interval with Ag85A-DNA <i>M. ulcerans</i> (white bars) or Ag85A-DNA <i>M. tuberculosis</i> (black bars).</p

Mycobacterial multiplication in mice infected with <i>M. ulcerans</i> strain 04-855.

<p>Mice infected three months after the last vaccination (protocol 1 using Gerbu adjuvant). Results are mean±SD of AFB/ml of 4 individual mice, expressed in Log₁₀. * p value as compared to mean log₁₀ value obtained in control DNA mice infected with <i>M. ulcerans.</i></p

Ag85 <i>M. ulcerans</i> specific total IgG, IgG1 and IgG2b antibody levels in B6 mice.

<p>Protein emulsified in Gerbu adjuvant. Data represent individual antibody levels (4 mice/group), mean value is indicated by the horizontal bars. * statistical analysis showing each group with a significant difference. For IgG1, groups c, d, f and g have a significative difference with the others groups. For IgG2b, only rec 85A Mu was significatively different from group a and e.</p

Evaluation of footpad size (A and C) and Survival curves (B and D) of vaccinated B6 mice after <i>M. ulcerans</i> infection with 10⁵ AFB in the right footpad.

<p>Experiment 2 using MPL-A as adjuvant and 6 weeks as rest between vaccination and infection. Mice were vaccinated with empty vector (* <i>n</i> = 9), with 0.2 mg of BCG (▵ <i>n</i> = 10), with Ag85-DNA from <i>M. ulcerans</i> (□ <i>n</i> = 9), with DNA encoding 85A from <i>M. tuberculosis</i> (○ <i>n</i> = 10), with Ag85A DNA-<i>M. ulcerans</i> boosted with the Ag85A protein from <i>M. ulcerans</i> (▪ <i>n</i> = 9), or with DNA encoding Ag85A boosted with Ag85 protein from <i>M. tuberculosis</i> (• <i>n</i> = 10).</p

Spleen cell IL-2 (A) and IFN-γ (B) responses to whole Ag85A- <i>M. ulcerans</i> and its synthetic peptides, as tested on a pool of six B6 mice vaccinated 3 times at three weeks interval with Ag85A-DNA <i>M. ulcerans</i> (white bars) or Ag85A-DNA <i>M. tuberculosis</i> (black bars).

<p>Graph in insert represents the cytokine production in response to partial Ag85A <i>M. ulcerans</i> aa 17–150. Unstimulated cells (grey bars). Data of insert represent mean±SD values of 3 mice tested individually.</p

Inhibition of proliferation of L929 fibroblasts upon treatment with PG-120, PG-119 and mycolactone A/B.

<p>Cell proliferation was measured after 0, 24, 48 and 72 h by determining the number of cells treated with 60 ng/ml of PG-120, PG-119 or mycolactone A/B. Mean values and standard deviations of triplicates are shown.</p

Comparison of LC₅₀ and IC₅₀ values of the mycolactone variants.

<p>Comparison of LC₅₀ and IC₅₀ values of the mycolactone variants.</p

Cytotoxicity of truncated mycolactone variants.

<p>Cells were treated for 48 hours with different concentrations of PG-119 or PG-120 and stained with annexin-V-FITC and PI. Flow cytometry was used to determine the annexin-positive (A⁺) and PI-positive (PI⁺) cell populations. Triplicate samples were analyzed and mean values as well as standard deviations are shown.</p