Fig 1.
Synthetic mycolactone derivatives used for the generation and detection of mycolactone specific antibody responses.
PG-203 (A) was conjugated to BSA via the amino group on its diethylene glycol-based linker and the carrier protein conjugate was used for the immunization of mice. PG-204 (C) and PG-183 (D) are biotinylated mycolactone derivatives used for analytical purposes. Unmodified synthetic mycolactone A/B (B) was used as effector molecule in cytotoxicity assays.
Fig 2.
Mice immunized with the adjuvanted PG-203-BSA conjugate developed a boostable anti-mycolactone IgG response.
Displayed are average anti-mycolactone IgG titers of three mice after the second and third immunization in comparison to pooled pre-immune sera. Titers were determined by ELISA using NeutrAvidin plates coated with PG-204.
Fig 3.
The anti-mycolactone mAbs JD5.1 to JD5.12 bind to PG-204, but not to PG-183.
The biotinylated mycolactone derivatives PG-204 and PG-183 were bound to NeutrAvidin plates and incubated with serial dilutions of the mAbs. Bound anti-mycolactone mAbs were detected using alkaline phosphatase-conjugated goat anti-mouse IgG antibodies.
Fig 4.
Effect of structural modifications of mycolactone on antibody binding.
Competition ELISAs were performed by coating microtiter plates with the mAbs and pre-incubation with serial dilutions of mycolactone A/B, natural mycolactone variants harboring modifications in the lower fatty acid acyl side chain (mycolactone C and mycolactone F), a truncated core molecule without O-linked lower side chain (PG-119) or derivatives containing substitutions in the upper side chain (PG-165, PG-157 and PG-182). Biotinylated PG-204 was subsequently added and its binding was detected by using alkaline phosphatase-conjugated streptavidin as reporter. Detailed results with all tested mycolactone derivatives are shown for mAb 5.1 as example.
Fig 5.
Two types of reactivity patterns.
All mAbs displayed a similar fine specificity pattern, except for mAbs 5.9 and 5.11, which, in contrast to the other mAbs, showed inhibition with PG-157, a derivative with a slightly extended upper side chain (the difference is depicted for the prototype mAbs JD5.1 and JD5.11).
Fig 6.
SPR analysis of anti-mycolactone mAbs.
Anti-mycolactone mAbs were captured on the chip surface by primary antibodies. The biotinylated mycolactone derivative PG-204 was then titrated over the surface and association and dissociation phases were monitored for 240 and 3600 s, respectively. Prototype titration binding curves for two selected mAbs (JD5.8 and JD5.10) are shown. Titration of both antibodies was performed up to 500 nM with a dilution factor of 2.
Fig 7.
Toxin neutralizing activity of anti-mycolactone mAbs.
L929 fibroblasts were incubated with 15 ng/ml (20 nM) synthetic mycolactone A/B and mAbs were added in different molar ratios of excess antibody versus mycolactone. After 48 h, the metabolic activity of fibroblasts was measured. The graph shows mean values across duplicate samples, and the error bars represent the standard deviation of three independent experiments. As control, the isotype-matched antibody JD4.1 was used.
Fig 8.
Inhibition of antibody mediated neutralization by addition of the non-toxic mycolactone core variant PG-119.
L929 cells were treated with a mix of 15 ng/ml (20 nM) synthetic mycolactone A/B and a 2.5 fold molar excess of the anti-mycolactone mAb JD5.1. Serial dilutions of PG-119 were added to the mix, starting with a PG-119 concentration of 40 ng/ml (86 nM). As control, the isotype-matched antibody JD4.1 was used. After 48 h, the metabolic activity of fibroblasts was measured and plotted against the concentration of PG-119. The graph shows mean values across duplicate samples, and the error bars represent the standard deviation of three independent experiments.