Characterization of antibody-protein interactions in Diphtheria Toxin
Research area: Structural Biology
Group leaders: Luca Varani
- Mattia Pedotti, TechnicianResearch Assistant
Status: In progress
Diphtheria is an acute infectious disease caused by the bacterial Diphtheria Toxin (DT). Although mass immunization has virtually eradicated diphtheria from the western world, the disease continues to be a serious health threat in regions like the former USSR, Asia and South America. In the 1990s, for instance, an epidemic caused approximately four thousand deaths in Russia even amongst formerly vaccinated individual, apparently due to a decline in adult immunity level. Beside the medical implications, diphtheria toxin has been extensively characterized at the biochemical level and represents a good model for the study of antibody-toxin interactions. Curiously, however, no structural information on DT-antibody complexes is available so far.
The Lanzavecchia group has isolated a number of human monoclonal antibodies with remarkably strong binding affinity for DT. Some of these antibodies are very potent neutralizers but, intriguingly, they are not those with the stronger binding affinity according to surface plasmon resonance (SPR) measurements. If not binding affinity, what are the determinants of efficient toxin neutralization?
DT is formed by three separated protein domains. ELISA, SPR and cross-competition experiments showed that the best neutralizing antibodies target different regions of the so-called receptor binding domain (figure 4). Perhaps unintuitively, however, we were able to show that the best antibodies do not inhibit interaction of DT with its cellular receptor. We are now trying to determine the mechanism of action of these antibodies through cellular and in vitro experiments. One possibility is that they might block the conformational change required for the activation of DT and subsequent toxicity.
We were able to characterize three groups of human antibodies that bind Diphtheria Toxin (DT) with similar affinity (nanomolar) but have different neutralizing efficacy. Antibodies against the catalytic domain (yellow in the figure) are the worst neutralizer in our panel. Antibodies against the receptor binding domain (red in the figure) show intermediate to high neutralization according to their binding site on this domain.